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G protein‐coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310. Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308, leading to a shift in ligand affinity from a high‐to‐low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1‐engaged conformations of β‐arrestin and subsequent alterations in signal transduction. Stimulation of the high‐affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2‐GRK pathway, resulting in a β‐arrestin‐bound low‐affinity state. This configuration, referred to as the "low‐LTB4‐induced complex," necessitates the finger loop region and the phosphoinositide‐binding motif of β‐arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low‐affinity BLT1 again binds to the ligand and triggers the generation of the low‐LTB4‐induced complex into a different form termed "high‐LTB4‐induced complex." This change is propelled by The308‐phosphorylation‐dependent basal phosphorylation by PKCs. Within the high‐LTB4‐induced complex, β‐arrestin adapts a unique configuration that involves additional N domain interaction to the low‐affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein β‐arrestins perform distinct functions. [ABSTRACT FROM AUTHOR]

Background: In this study, we aimed to investigate the potential of miR‐19a as a biomarker of OSCC and its underlying molecular mechanisms. Methods: We collected serum and saliva samples from 66 OSCC patients and 66 healthy control subjects. Real‐time PCR analysis, bioinformatic analysis and luciferase assays were performed to establish a potential signaling pathway of miR‐19a/GRK6/GPCRs/PKC. Flowcytometry and Transwell assays were performed to observe the changes in cell apoptosis, metastasis and invasion. Results: We found that miR‐19a, GPR39 mRNA and PKC mRNA were upregulated while GRK6 mRNA was downregulated in the serum and saliva samples collected from OSCC patients. Moreover, in silico analysis confirmed a potential binding site of miR‐19a on the 3′UTR of GRK6 mRNA, and the subsequent luciferase assays confirmed the molecular binding between GRK6 and miR‐19a. We further identified that the over‐expression of miR‐19a could regulate the signaling between GRK6, GPR39 and PKC via the signaling pathway of miR‐19a/GRK6/GPR39/PKC, which accordingly resulted in suppressed cell apoptosis and promoted cell migration and invasion. Conclusion: Collectively, the findings of our study propose that miR‐19a is a crucial mediator in the advancement of OSCC, offering a potential avenue for the development of innovative therapeutic interventions aimed at regulating GRK6 and its downstream signaling pathways. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the etiological pathogen of coronavirus disease 2019 (COVID‐19), a highly contagious disease, spreading quickly and threatening global public health. The symptoms of COVID‐19 vary from mild reactions to severe respiratory distress or even fatal outcomes probably due to the different status of host immunity against the virus. Here in the study, we unveiled plasma proteomic signatures and transcriptional patterns of peripheral blood mononuclear cells (PBMCs) using blood samples of 10 COVID‐19 patients with different severity. Through systemic analysis, α‐defensin‐1 (DEFA1) was identified to be elevated in both plasma and PBMCs, and correlated with disease severity and stages. In vitro study demonstrated that DEFA1 was secreted from immunocytes and suppressed SARS‐CoV‐2 infection of both original and mutated strains with dose dependency. By using sequencing data, we discovered that DEFA1 was activated in monocytes through NF‐κB signaling pathway after infection, and secreted into circulation to perturb SARS‐CoV‐2 infection by interfering protein kinase C expression. It worked mainly during virus replication instead of entry in host cells. Together, the anti‐SARS‐CoV‐2 mechanism of DEFA1 has unveiled a corner of how innate immunity is against SARS‐CoV‐2 and explored its clinical potential in disease prognosis and therapeutic intervention. [ABSTRACT FROM AUTHOR]

FTY720P, an analogue of sphingosine 1‐phosphate, has emerged lately as a potential causative agent of inflammatory bowel disease, in which electrolytes movements driven by the sodium gradient established by the Na+/K+ ATPase are altered. We showed previously in Caco‐2 cells, a 50% FTY720P‐induced decrease in the ATPase activity, mediated via S1PR2 and PGE2. This work aims at delineating the mechanism underlying PGE2 release and at investigating if the ATPase inhibition is due to changes in its abundance. The activity of the ATPase and the localization of a GFP‐tagged Na+/K+‐ATPase α1‐subunit were assessed in cells treated with 7.5 nM FTY720P. The involvement of ERK, p38 MAPK, PKC, and PI3K was studied in cells treated with 7.5 nM FTY720P or 1 nM PGE2 in presence of their inhibitors, or by determining changes in the protein expression of their activated phosphorylated forms. Imaging data showed ∼30% reduction in the GFP‐tagged Na+/K+ ATPase at the plasma membrane. Both FTY720P and PGE2 showed, respectively, 50% and 60% reduction in ATPase activity that disappeared when p38 MAPK, PKC, and PI3K were inhibited individually but not with ERK inhibition. The effect of FTY720P was imitated by PMA, an activator of PKC. Western blotting revealed inhibition of ERK by FTY720P. It was concluded that FTY720P, through activation of S1PR2, downregulates the Na+/K+ ATPase by inhibiting ERK, which in turn activates p38 MAPK leading to the sequential activation of PKC and PI3K, PGE2 release, and a decrease in the Na+/K+ ATPase activity and membrane abundance. We showed previously in Caco‐2 cells, a significant FTY720P‐induced decrease in the Na+/K+ ATPase activity, mediated via PGE2. Fluorescence imaging of GFP‐tagged Na+/K+ ATPase showed that the reduced ATPase activity is due to a lower membrane abundance and is mediated through activation of S1PR2, which inhibits ERK. The latter activates p38 MAPK leading to the sequential activation of PKC, PI3K, PGE2 release, and downregulation of the Na+/K+ ATPase. [ABSTRACT FROM AUTHOR]

Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin‐stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G‐protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down‐regulation increases BK receptor desensitization in real‐time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2‐sensitive G‐protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization. [ABSTRACT FROM AUTHOR]

Differential mechanisms of insulin resistance in muscle fibre types Jani and colleagues were able to supply new insight into the development of insulin resistance in female skeletal muscle, and how this may differ between fibre types. Keywords: ceramides; DAGs; inflammation; insulin resistance; muscle fibres; PKC; skeletal muscle EN ceramides DAGs inflammation insulin resistance muscle fibres PKC skeletal muscle 2241 2243 3 06/19/23 20230615 NES 230615 Skeletal muscle insulin resistance It is well recognized that the inclusion of females in metabolic research is limited, meaning that much of our current understanding of physiological processes stems primarily from research using males and may therefore not be generalizable. One size does not fit all: insight into skeletal muscle insulin resistance in female rats fed a high-fat sucrose-enriched diet. [Extracted from the article]

In the present study, the participation of protein kinase C (PKC) signalling in prothoracicotropic hormone (PTTH)‐stimulated ecdysteroidogenesis in Bombyx prothoracic glands (PGs) is demonstrated and characterized. PTTH stimulated phosphorylation of a 37‐kDa protein in Bombyx PGs both in vitro and in vivo, as recognized by a PKC substrate antibody. Treatment with either A23187 or thapsigargin also stimulated this 37‐kDa protein phosphorylation. PTTH‐stimulated phosphorylation of the 37‐kDa protein was markedly attenuated in the absence of Ca2+. The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH‐stimulated phosphorylation of this protein, indicating the involvement of Ca2+ and PLC. A mitogen‐activated protein kinase/extracellular signal‐regulated kinase (ERK) kinase (MEK) inhibitor (U0126), a phosphoinositide 3‐kinase (PI3K) inhibitor (LY294002) and a chemical activator of adenosine 5′‐monophosphate‐activated protein kinase (AMPK) (5‐aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside) did not affect PTTH‐stimulated phosphorylation of the 37‐kDa protein, implying that ERK and PI3K/AMPK are not the upstream signalling pathways for PKC‐dependent protein phosphorylation. The mitochondrial oxidative phosphorylation inhibitors (the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone and diphenylene iodonium) inhibited PTTH‐stimulated phosphorylation of the 37‐kDa protein, indicating its redox regulation. Treatment with PKC inhibitors (either calphostin C, chelerythrine C or rottlerin) reduced PTTH‐stimulated phosphorylation of the 37‐kDa protein. PTTH‐stimulated ecdysteroidogenesis was also inhibited by treatment with rottlerin, thus further confirming participation of PKC‐dependent phosphorylation in PTTH signalling. From these results, we demonstrated that redox‐regulated PTTH‐stimulated PKC signalling is involved in ecdysteroid secretion in Bombyx PGs. [ABSTRACT FROM AUTHOR]

Annexin A2 (ANXA2) is a multifunctional protein expressed in nearly all human tissues and cell types, playing a role in various signaling pathways. It is subjected to phosphorylation, but no specific protein phosphatase has been identified in its posttranslational regulation yet. Using pull‐down assay followed by liquid chromatography–mass spectrometry analysis we found that ANXA2 interacts with TIMAP (TGF‐beta‐inhibited membrane‐associated protein) in pulmonary artery endothelial cells. TIMAP is highly expressed in endothelial cells, where it acts as a regulatory and targeting subunit of protein phosphatase 1 (PP1). TIMAP plays an important role in the regulation of the endothelial barrier maintenance through the dephosphorylation of its several substrate proteins. In the present work, phosphorylation of Ser25 side chain in ANXA2 by protein kinase C (PKC) was shown both in vivo and in vitro. Phosphorylation level of ANXA2 at Ser25 increased greatly by inhibition of PP1 and by depletion of its regulatory subunit, TIMAP, implying a role of this PP1 holoenzyme in the dephosphorylation of ANXA2. Immunofluorescence staining and subcellular fractionations revealed a diffuse subcellular localization for the endogenous ANXA2, but phospho‐Ser25 ANXA2 was mainly detected in the membrane. ANXA2 depletion lowered the basal endothelial barrier and inhibited cell migration, but had no significant effect on cell proliferation or viability. ANXA2 depleted cells failed to respond to PMA treatment, indicating an intimately involvement of phospho‐ANXA2 in PKC signaling. Moreover, phosphorylation of ANXA2 disrupted its interaction with S100A10 suggesting a phosphorylation dependent multiple regulatory role of ANXA2 in endothelial cells. Our results demonstrate the pivotal role of PKC‐ANXA2‐PP1 pathway in endothelial cell signaling, especially in barrier function and cell migration. [ABSTRACT FROM AUTHOR]

It is well known that phosphorylation of extracellular signal‐regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)‐stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage‐ and dose‐dependent. The absence of Ca2+ reduced PTTH‐stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH‐stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH‐regulated RSK phosphorylation, indicating its redox regulation. A mitogen‐activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3‐kinase (PI3K) inhibitor, LY294002, decreased PTTH‐stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH‐stimulated RSK phosphorylation, and a PKC activator, phorbol 12‐myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI‐D1870, a specific RSK inhibitor, partly prevented PTTH‐stimulated RSK phosphorylation and significantly inhibited PTTH‐stimulated ecdysteroid secretion, indicating that PTTH‐stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH‐stimulated ecdysteroidogenesis. [ABSTRACT FROM AUTHOR]

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer's disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans. [ABSTRACT FROM AUTHOR]

Neuromuscular junctions (NMJ) regulate cholinergic exocytosis through the M1 and M2 muscarinic acetylcholine autoreceptors (mAChR), involving the crosstalk between receptors and downstream pathways. Protein kinase C (PKC) regulates neurotransmission but how it associates with the mAChRs remains unknown. Here, we investigate whether mAChRs recruit the classical PKCβI and the novel PKCε isoforms and modulate their priming by PDK1, translocation and activity on neurosecretion targets. We show that each M1 and M2 mAChR activates the master kinase PDK1 and promotes a particular priming of the presynaptic PKCβI and ε isoforms. M1 recruits both primed‐PKCs to the membrane and promotes Munc18‐1, SNAP‐25, and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA‐dependent pathway, which inhibits Munc18‐1 synthesis and PKC phosphorylation. In summary, our results discover a co‐dependent balance between muscarinic autoreceptors which orchestrates the presynaptic PKC and their action on ACh release SNARE‐SM mechanism. Altogether, this molecular signaling explains previous functional studies at the NMJ and guide toward potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Meprin ß is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin ß including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin ß is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin ß including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin ß with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin ß from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin ß was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin ß phosphorylation and identified the involvement of PKC-a and PKC-ß. As a result of phosphorylation, the meprin ß activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin ß. [ABSTRACT FROM AUTHOR]

Background: Heat shock protein 90 (Hsp90) signaling pathways participate in protein phosphorylation during sperm capacitation. However, the underlying mechanism is largely unknown. Objective: The aim of this study was to explore the interaction between Hsp90 and its co‐chaperone protein, cell division cycle protein Cdc37 (Cdc37), in human spermatozoa. Materials and methods: We examined the effects of H‐89 (a protein kinase A [PKA] inhibitor) and Go6983 (a protein kinase C [PKC] inhibitor) on the phosphorylation of serine, threonine, and tyrosine residues in Hsp90; the effect of 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG, Hsp90 inhibitor) on Y416‐Src phosphorylation; and the effects of 17‐AAG and geldanamycin on threonine phosphorylation during human sperm capacitation. Results: Hsp90 co‐localized and interacted with Cdc37. During human sperm capacitation, Hsp90 phosphorylation at serine, threonine, and tyrosine residues was inhibited by H‐89 and Go6983. In addition, phosphorylation of residue Y416 in the tyrosine kinase Src (its active site) was inhibited by 17‐AAG, and the threonine phosphorylation levels of some proteins were decreased by 17‐AAG and geldanamycin. Discussion and conclusion: Taken together, our data showed that the interaction of Hsp90 with Cdc37 regulates total protein threonine phosphorylation and Src phosphorylation via its serine, threonine, and tyrosine phosphorylation, which are controlled by PKA and PKC during human sperm capacitation. The results of this study help understand the mechanism underlying Hsp90 regulation of sperm function. [ABSTRACT FROM AUTHOR]

Melanocyte survival is mediated by diverse signaling pathways. However, the molecular mechanisms they use and molecules that they target are incompletely understood. Here, we show that melanocyte survival is mediated by diverse, nonredundant signaling pathways, including ERK1/2, AKT, PKA, and PKC. Each of these pathways is exerting prosurvival effects by phosphorylating the BAD. While Ser112‐BAD phosphorylation is regulated by pERK, pPKA and pPKC, Ser136 and Ser155 phosphorylation are exclusively controlled by pAKT and pPKA, respectively. Inhibition of these pathways individually resulted in only modest apoptosis; however, most significant apoptosis, as a result of BAD dephosphorylation, was seen when all pathways were inhibited concurrently. BAD phosphorylation was essential for survival of melanocytes as cells expressing phosphorylation‐deficient BAD were not rescued by any of the identified pathway. Furthermore, melanocytes became insensitive to kinase inhibitor‐induced apoptosis when BAD expression was knocked down by BAD‐shRNA. Overexpression of BAD in melanocytes stimulated faster apoptosis in response to kinase inhibitors. Taken together, our results show that BAD is acting as a convergence point for diverse survival pathways in melanocytes. Understanding the molecular mechanisms of melanocyte survival provides fundamental new insights into physiological mechanisms involved in the development of various melanocyte pathologies such as melanoma and vitiligo. [ABSTRACT FROM AUTHOR]

Key points: In vascular smooth muscle cells (VSMCs), activation of Ca2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP2 and TRPC1 and activation of TRPC1‐based SOCs by PIP2 required PKCδ.These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP2‐mediated channel opening. In vascular smooth muscle cells (VMSCs), stimulation of Ca2+‐permeable canonical transient receptor potential channel 1 (TRPC1)‐based store‐operated channels (SOCs) mediates Ca2+ entry pathways that regulate cell contraction, proliferation and migration, which are processes associated with vascular disease. It is therefore important to understand how TRPC1‐based SOCs are activated. Stimulation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, with store‐operated PKC‐dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). Experimental protocols used to activate TRPC1‐based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+‐insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence, the present study examined whether a novel PKC isoform(s) is involved in activating TRPC1‐based SOCs in contractile rat mesenteric artery VSMCs. Store‐operated whole‐cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1‐based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store‐operated PIP2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP2‐mediated activation of TRPC1 currents. These results identify the involvement of PKCδ in stimulation of TRPC1‐based SOCs and highlight that store‐operated PKCδ activity is obligatory for channel opening by PIP2, the probable activating ligand. Key points: In vascular smooth muscle cells (VSMCs), activation of Ca2+‐permeable store‐operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediates Ca2+ entry pathways that regulate contraction, proliferation and migration, which are processes associated with vascular disease.Activation of TRPC1‐based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5‐bisphosphate (PIP2). We investigated the identity of the PKC isoform involved in activating TRPC1‐based SOCs in rat mesenteric artery VSMCs.TRPC1‐based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ‐dependent phosphorylation of TRPC1. Furthermore, generation of store‐operated interactions between PIP2 and TRPC1 and activation of TRPC1‐based SOCs by PIP2 required PKCδ.These findings reveal that PKCδ activity has an obligatory role in activating TRPC1‐based SOCs, through regulating PIP2‐mediated channel opening. [ABSTRACT FROM AUTHOR]

Vestibular compensation (VC) refers to a behavioral recovery process in which firing rates of bilateral vestibular nuclei neurons are rebalanced. Our study aimed to investigate the underlying mechanism by which miR‐219a‐5p regulates Ca2+/calmodulin–dependent protein kinase II γ isoform (CaMKIIγ) and protein kinase C (PKC) in VC. A unilateral vestibular deafferentation rat model was established by unilateral labyrinthectomy (UL), after which VC was evaluated in rats with UL‐induced vertigo‐like behavior by measuring vestibular defect behavior and performing rotarod tests, as well as by BrdU immunohistochemistry on medial vestibular nuclei. We found that miR‐219a‐5p was increased while CaMKIIγ was decreased during VC in the medial vestibular nucleus of rats that had undergone UL. Next, gain‐ and loss‐of‐function assays were conducted to evaluate the effects of miR‐219a‐5p and CaMKIIγ on the vestibular defect behaviors and VC, the results of which suggested that in rats after UL overexpression of CaMKIIγ inhibited VC, while overexpression of miR‐219a‐5p facilitated VC. A dual‐luciferase reporter gene assay identified that miR‐219a‐5p targeted CaMKIIγ. This led to additional experiments showing that miR‐219a‐5p aptomir expression downregulated CaMKIIγ in cortical cells with a concomitant increase in PKC expression, which were verified further in vivo. In summary, in rats with acute vertigo, miR‐219a‐5p overexpression inhibits CaMKIIγ and elevates PKC, thereby facilitating VC. Our study offers possible targets for further evaluation as treatment of acute vertigo in humans. [ABSTRACT FROM AUTHOR]

We sought to determine the mechanism by which angiotensin II (AngII) inhibits isoproterenol induced increase in adenylate cyclase (AC) activity and cyclic adenosine monophosphate (cAMP) production in bovine pulmonary artery smooth muscle cells (BPASMCs). Treatment with AngII stimulates protein kinase C‐ζ (PKC‐ζ), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and PKC‐α activities, and also inhibits isoproterenol induced increase in AC activity and cAMP production in the cells. Pertussis toxin pretreatment eliminates AngII caused inhibition of isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ, NADPH oxidase, and PKC‐α activities. Treatment of the cells with AngII increases α2 isoform of Gi (Giα2) phosphorylation; while pretreatment with chemical and genetic inhibitors of PKC‐ζ and NADPH oxidase attenuate AngII induced increase in PKC‐α activity and Giα2 phosphorylation, and also reverse AngII caused inhibition of isoproterenol induced increase in AC activity. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐α attenuate AngII induced increase in Giα2 phosphorylation and inhibits isoproterenol induced increase in AC activity without a discernible change in PKC‐ζ and NADPH oxidase activities. Overall, PKCζ‐NADPH oxidase‐PKCα signaling axis plays a crucial role in Giα2 phosphorylation resulting in AngII‐mediated inhibition of isoproterenol induced increase in AC activity in BPASMCs. [ABSTRACT FROM AUTHOR]

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)‐induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact‐mediated functions, Sema6A may have regulated, long‐range, forward‐signaling capacity. [ABSTRACT FROM AUTHOR]

Key points: Acute hyperglycaemia at the time of a heart attack worsens the outcome for the patient.Acute hyperglycaemia is not limited to diabetic patients and can be due to a stress response in non‐diabetics.This study suggests that the damaging cardiac effects of hyperglycaemia can be reversed by selective PKC inhibition.If PKCα/β isoforms are inhibited, then high glucose itself becomes protective against ischaemic damage.Selective PKC inhibition may therefore be a useful therapeutic tool to limit the damage that can occur during a heart attack by stress-induced hyperglycaemia. Hyperglycaemia has a powerful association with adverse prognosis for patients with acute coronary syndromes (ACS). Previous work shows that high glucose prevents ischaemic preconditioning and causes electrical and mechanical disruption via protein kinase C α/β (PKCα/β) activation. The present study aimed to: (i) determine whether the adverse clinical association of hyperglycaemia in ACS can be replicated in preclinical cellular models of ACS and (ii) determine the importance of PKCα/β activation to the deleterious effect of glucose. Freshly isolated rat, guinea pig or rabbit cardiomyocytes were exposed to simulated ischaemia after incubation in the presence of normal (5 mm) or high (20 mm) glucose in the absence or presence of small molecule or tat‐peptide‐linked PKCαβ inhibitors. In each of the four conditions, the following hallmarks of cardioprotection were recorded using electrophysiology or fluorescence imaging: cardiomyocyte contraction and survival, action potential stability and time to failure, intracellular calcium and ATP, mitochondrial depolarization, ischaemia‐sensitive leak current, and time to Kir6.2 opening. High glucose alone resulted in decreased cardiomyocyte contraction and survival; however, it also imparted cardioprotection in the presence of PKCα/β inhibitors. This cardioprotective phenotype displayed improvements in all of the measured parameters and decreased myocardium damage during whole heart coronary ligation experiments. High glucose is deleterious to cellular and whole‐heart models of simulated ischaemia, in keeping with the clinical association of hyperglycaemia with an adverse outcome in ACS. PKCαβ inhibition revealed high glucose to show a cardioprotective phenotype in this setting. The results of the present study suggest the potential for the therapeutic application of PKCαβ inhibition in ACS associated with hyperglycaemia. Key points: Acute hyperglycaemia at the time of a heart attack worsens the outcome for the patient.Acute hyperglycaemia is not limited to diabetic patients and can be due to a stress response in non‐diabetics.This study suggests that the damaging cardiac effects of hyperglycaemia can be reversed by selective PKC inhibition.If PKCα/β isoforms are inhibited, then high glucose itself becomes protective against ischaemic damage.Selective PKC inhibition may therefore be a useful therapeutic tool to limit the damage that can occur during a heart attack by stress-induced hyperglycaemia. [ABSTRACT FROM AUTHOR]

For many decades, kinases have predominantly been characterized as oncogenes and drivers of tumorigenesis, because activating mutations in kinases occur in cancer with high frequency. The oncogenic functions of kinases relate to their roles as growth factor receptors and as critical mediators of mitogen‐activated pathways. Indeed, some of the most promising cancer therapeutic agents are kinase inhibitors. However, cancer genomics studies, especially screens that utilize high‐throughput identification of loss‐of‐function somatic mutations, are beginning to shed light on a widespread role for kinases as tumor suppressors. The initial characterization of tumor‐suppressing kinases— in particular members of the protein kinase C (PKC) family, MKK4 of the mitogen‐activated protein kinase kinase family, and DAPK3 of the death‐associated protein kinase family— laid the foundation for bioinformatic approaches that enable the identification of other tumor‐suppressing kinases. In this review, we discuss the important role that kinases play as tumor suppressors, using several examples to illustrate the history of their discovery and highlight the modern approaches that presently aid in the identification of tumor‐suppressing kinases. © 2018 IUBMB Life, 71(6):738–748, 2019 [ABSTRACT FROM AUTHOR]

Summary: Non‐alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance (IR). Resveratrol (RES) a potent hypolipidemic dietary polyphenol has been identified for its ability to prevent hepatic steatosis and hepatic IR in high‐fat diet (HFD)‐fed murine models of NAFLD. In the present study, we have carried an in vivo animal experiment to identify a novel mechanism for RES protective action. Sub‐chronic (45 days) RES pretreatment in 3 days HFD‐fed adult Wistar rats prevented early hepatic IR through inhibiting PKC/JNK activation; decreasing p‐IRS (Ser307) and increasing p‐IRS(Tyr612), p‐Akt(Ser473) and p‐GSK3(Ser9). These effects of RES were associated with reduced expression of acyl‐CoA:glycerol‐sn‐3‐phosphate acyltransferase (GPAT‐1) and diacylglycerol:acyl‐CoA acyltransferase (DGAT2), two critical enzymes in the glycerol‐3‐phosphate pathway for de novo triglycerides synthesis. These data indicate that RES protects against NAFLD, initially, by inhibiting the early development of hepatic IR. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER)-plasma membrane (PM) contacts are dynamic structures with important roles in the regulation of calcium (Ca2+) and lipid homeostasis. The extended synaptotagmins (E-Syts) are ER-localized lipid transport proteins that interact with PM phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. E-Syts bidirectionally transfer glycerolipids, including diacylglycerol (DAG), between the 2 juxtaposed membranes, but the biologic significance of this transport is still unclear. Using insulin-secreting cells and live-cell imaging, we now show that Ca2+-triggered exocytosis of insulin granules is followed, in sequence, by PM DAG formation and E-Syt1 recruitment. E-Syt1 counteracted the depolarization-induced DAG formation through a mechanism that required both voltage-dependent Ca2+ influx and Ca2+ release from the ER. E-Syt1 knockdown resulted in prolonged accumulation of DAG in the PM, resulting in increased glucose-stimulated insulin secretion. We conclude that Ca2+-triggered exocytosis is temporally coupled to Ca2+-triggered E-Syt1 PM recruitment and removal of DAG to negatively regulate the same process. [ABSTRACT FROM AUTHOR]

Atopic dermatitis is a multifactorial skin disease characterised by chronic and relapsing inflammation whose pathogenesis is incompletely understood. We found that the expression of TGFβR1 and the activation of SMAD2, RhoA, JNK, PKC‐βII/δ and c‐Src were upregulated in the infiltrated inflammatory cells, fibroblasts and vasculatures in the dermis and epidermis. In addition, increases in the expression of TGFβR1 and phosphorylation levels of JNK and c‐Src were positively correlated with the inflammatory progression of atopic dermatitis severity. [ABSTRACT FROM AUTHOR]

Although our previous studies have demonstrated both protein kinase C (PKC) and GluN2B-containing N-methyl-d-aspartate receptor (GluN2B-NMDAR) play crucial roles in morphine-associated learning and memory, the relationship between them remains unexplored. In this study, we validated the enhanced PKC and membrane GluN2B protein expression in the hippocampal CA1 after morphine conditioned place preference (CPP) expression in rats. Interestingly, we also found that phosphorylation of SNAP25 at Ser187 (pSer187-SNAP25), a PKC-activated target, was significantly increased following morphine CPP expression. Blocking the pSer187-SNAP25 by intra-CA1 injection of an interfering peptide impaired morphine CPP expression and accompanied by the reduced ratio of GluN2B membrane/total in the CA1. In addition, intra-CA1 blockade of pSer187-SNAP25 did not affect natural learning and memory process as evidenced by intact sucrose-induced CPP expression and normal locomotor activity in rats. Therefore, our results reveal that enhanced pSer187-SNAP25 by PKC recruits GluN2B-NMDAR to the membrane surface in the hippocampal CA1 and mediates context-induced addiction memory retrieval. Our findings in this study fill in the missing link and provide better understanding of the molecular mechanisms involved in morphine-associated contextual memory retrieval. [ABSTRACT FROM AUTHOR]

Abstract: Background: Muscarinic acetylcholine receptor (mAChR) activation is an important factor to enhance the motility of gastrointestinal (GI) smooth muscle. Large conductance Ca2+‐activated potassium (BK) channels are widely expressed in GI smooth muscle. Roles of BK in carbachol (a mAChR agonist) induced enhancement of GI motility and the molecular mechanisms remains unknown and were investigated in this study. Methods: Colonic smooth muscle (CSM) strip was perfused to record motility in vitro. The patch‐clamp technique was used to record BK currents. RT‐PCR was used to detect the expression of BK channels in rat CSM tissues. Two different types BK channels were constructed in HEK293 cells to investigate the regulation mechanism. Paired t tests were set with a P < .05 regarded as significant. Key Results: Carbachol enhanced CSM contraction through M3 receptor (M3R) were attenuated by IbTX, an inhibitor of BK. Carbachol inhibited BK currents in CSM cells and Go6983, an inhibitor of protein kinase C (PKC), reversed the effect. PKC activator, phorbol 12‐myristate 13‐acetate (PMA), inhibited BK currents. Two types of BK channels (ZERO‐BK and STREX‐BK) were detected in CSM. ZERO‐ but not STREX‐BK channels expressed in HEK293 cells were inhibited by PMA. Conclusion: Our results provide strong evidence that inhibition of ZERO‐BK but not STREX‐BK channels via PKC pathway is involved in the enhancement of CSM motility by mAChR activation. Besides the activation of BK by an increase in intracellular calcium, inhibition of BK played an important role in GI motility regulation during mAChR activation. [ABSTRACT FROM AUTHOR]

Annexins, a family of highly conserved calcium- and phospholipid-binding proteins, play important roles in a wide range of physiologic functions. Among the 12 known annexins in humans, annexin A2 (AnxA2) is one of the most extensively studied and has been implicated in various human diseases. AnxA2 can exist as a monomer or a heterotetrameric complex with S100A10 (P11) and plays a critical role in many cellular processes, including exocytosis, endocytosis, and membrane organization. At the endothelial cell surface, the (AnxA2⋅P11)2 tetramer--acting as a coreceptor for plasminogen and tissue plasminogen activator (tPA)--accelerates tPA-dependent activation of the fibrinolytic protease, plasmin, the enzyme that is responsible for thrombus dissolution and the degradation of fibrin. This study demonstrates that EPAC1 (exchange proteins directly activated by cAMP isoform 1) interacts with AnxA2 and regulates its biologic functions by modulating its membrane translocation in endothelial cells. By using genetic and pharmacologic approaches, we demonstrate that EPAC1--acting via the PLCe-PKC pathway--inhibits AnxA2 surface translocation and plasminogen activation. These results suggest that EPAC1 plays a role in the regulation of fibrinolysis in endothelial cells and may represent a novel therapeutic target for disorders of fibrinolysis. [ABSTRACT FROM AUTHOR]

PKC-potentiated phosphorylation-dependent inhibitory protein of protein phosphatase 1 (CPI-17), an endogenous myosin phosphatase inhibitory protein, is considered a key molecule for Ca2+ sensitization of the contractile apparatus. Here, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 to generate CPI-17-deficient [knockout (KO)] and threonine 38 (T38)-phospho-resistant mice [threonine mutant into alanine (TA)], and then effects of CPI-17 on vascular contractility in vitro and mean blood pressure (MBP) in vivo were investigated. In isolated thoracic aorta, phorbol 12, 13-dibutyrate induced a sustained contraction of wild-type (WT) mice, whereas no contraction showed from TA or KO mice. A high concentration of KCl solution-induced contraction was not different between transgenic and WT mice. In contrast, phenylephrine (PE)-induced contractions in both mutant strains were significantly smaller than those of WT mice in association with a low level of myosin phosphorylation, suggesting that at least part of PE-induced contraction is regulated by phosphorylation of CPI-17 at T38. Finally, the physiologic role of CPI-17 in the regulation of blood pressure was investigated using radio telemetry. MBP was decreased significantly in both transgenic mice, even with a compensatory increase in heart rate. In summary, we generated KO and constitutively phospho-resistant mouse models of CPI-17 for the first time. p-CPI-17 at T38, possibly by PKC, could be important to maintain vascular contractility and blood pressure in vivo. [ABSTRACT FROM AUTHOR]

Nociceptors are receptors specifically involved in detecting a tissue damage and transducing it in an electrical signal. Nociceptor activation provoked by any kind of acute lesion is related to the release of several mediators of inflammation, within the framework of a process defined as "peripheral sensitization." This results in an exaggerated response to the painful stimulus, clinically defined as "primary hyperalgesia." The concept of "neuroplasticity" may explain the adaptive mechanisms carried out by the Nervous System in relation to a "harmful" damage; also, neuroplasticity mechanisms are also fundamental for rehabilitative intervention protocols. Here we review several studies that addressed the role of different receptors and ionic channels discovered on nociceptor surface and their role in pain perception. The changes in expression, distribution, and functioning of receptors and ionic channels are thought to be a part of the neuroplasticity property, through which the Nervous System constantly adapts to external stimuli. Moreover, some of the reviewed mediators are also been associated to "central sensitization," a process that results in pain chronicization when the painful stimulation is particularly prolonged or intense, and lastly leads to the memorization of the uncomfortable painful perception. [ABSTRACT FROM AUTHOR]

A diagnostic criterion for drug addiction, persistent drug‐craving continues to be the most treatment‐resistant aspect of addiction that maintains the chronic, relapsing, nature of this disease. Despite the high prevalence of psychomotor stimulant addiction, there currently exists no FDA‐approved medication for craving reduction. In good part, this reflects our lack of understanding of the neurobiological underpinnings of drug‐craving. In humans, cue‐elicited drug‐craving is associated with the hyperexcitability of prefrontal cortical regions. Rodent models of cocaine addiction indicate that a history of excessive cocaine‐taking impacts excitatory glutamate signaling within the prefrontal cortex to drive drug‐seeking behavior during protracted withdrawal. This review summarizes evidence that the capacity of cocaine‐associated cues to augment craving in highly drug‐experienced rats relates to a withdrawal‐dependent incubation of glutamate release within prelimbic cortex. We discuss how stimulation of mGlu1/5 receptors increases the activational state of both canonical and noncanonical intracellular signaling pathways and present a theoretical molecular model in which the activation of several kinase effectors, including protein kinase C, extracellular signal‐regulated kinase and phosphoinositide 3‐kinase (PI3K) might lead to receptor desensitization to account for persistent cocaine‐craving during protracted withdrawal. Finally, this review discusses the potential for existing, FDA‐approved, pharmacotherapeutic agents that target kinase function as a novel approach to craving intervention in cocaine addiction. [ABSTRACT FROM AUTHOR]

Taurolithocholate (TLC) produces cholestasis by inhibiting biliary solute secretion in part by retrieving MRP2 from the plasma membrane (PM). Tauroursodeoxycholate (TUDC) and cAMP reverse TLC-induced cholestasis by inhibiting TLC-induced retrieval of MRP2. However, cellular mechanisms for this reversal are incompletely understood. Recently, we reported that TLC decreases PM-MRP2 by activating PKCε followed by phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS). Thus, cAMP and TUDC may reverse TLC-induced cholestasis by inhibiting the TLC/PKCε/ MARCKS phosphorylation pathway. We tested this hypothesis by determining whether TUDC and/or cAMP inhibit TLC-induced activation of PKCε and phosphorylation of MARCKS. Studies were conducted in HuH-NTCP cell line and rat hepatocytes. Activation of PKCε was determined from the translocation of PKCε to PM using a biotinylation method. Phosphorylation of MARCKS was determined by immunoblotting with a phospho-MARCKS antibody. TLC, but not cAMP and TUDC, activated PKCε and increased MARCKS phosphorylation in HuH-NTCP as well in rat hepatocytes. Treatment with TUDC or cAMP inhibited TLC-induced activation of PKCε and increases in MARCKS phosphorylation in both cell types. Based on these results, we conclude that the reversal of TLC-induced cholestasis by cAMP and TUDC involves, at least in part, inhibition of TLC-mediated activation of the PKCε/MARCKS phosphorylation pathway. [ABSTRACT FROM AUTHOR]

Synaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate ( AMPA) receptors are essential mediators of neurotransmission in the central nervous system. Shisa9/cysteine-knot AMPAR modulating protein 44 ( CKAMP44) is a transmembrane protein recently found to be present in AMPA receptor-associated protein complexes. Here, we show that the cytosolic tail of Shisa9/ CKAMP44 interacts with multiple scaffold proteins that are important for regulating synaptic plasticity in central neurons. We focussed on the interaction with the scaffold protein PICK1, which facilitates the formation of a tripartite complex with the protein kinase C ( PKC) and thereby regulates phosphorylation of Shisa9/ CKAMP44 C-terminal residues. This work has implications for our understanding of how PICK1 modulates AMPAR-mediated transmission and plasticity and also highlights a novel function of PKC. [ABSTRACT FROM AUTHOR]

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)-induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase-2 ( ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross-clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout ( ALDH2−/−) and wild-type ( WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C ( PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin-related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N-acetylcysteine ( NAC) or PKC-δ sh RNA treatment on glycogen synthase kinase-3β ( GSK-3β) activity and mitochondrial permeability transition pore ( mPTP) opening were also detected. The results showed that ALDH2−/− mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC-ε translocation was lower in ALDH2−/− mice than in WT mice, and PKC-δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species ( ROS) level, which could be reversed by NAC pre-treatment under I/R injury. In addition, PKC-ε inhibition caused activation of caspase9, caspase3 and Drp1Ser616 in response to I/R stress. Importantly, expression of phosphorylated GSK-3β (inactive form) was lower in ALDH2−/− mice than in WT mice, and both were increased by NAC pre-treatment. I/R-induced mitochondrial translocation of GSK-3β was inhibited by PKC-δ sh RNA or NAC pre-treatment. In addition, mitochondrial membrane potential (∆Ψm) was reduced in ALDH2−/− mice after I/R, which was partly reversed by the GSK-3β inhibitor ( SB216763) or PKC-δ sh RNA. Collectively, our data provide the evidence that abnormal PKC-ε/ PKC-δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK-3β-dependent mPTP opening, which results in mitochondrial injury-triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2−/− mice following I/R stress. [ABSTRACT FROM AUTHOR]

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N- and C-terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named 'gigantic M1 (GM1)' and 'gigantic displaced M1 (GDM1).' Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin-ir (0.63-0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin-ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin-ir RGCs, which most likely are involved in different functions. [ABSTRACT FROM AUTHOR]

CUB domain-containing protein-1 ( CDCP1) is a trans-membrane protein predominantly expressed in various cancer cells and involved in tumor progression. CDCP1 is phosphorylated at tyrosine residues in the intracellular domain by Src family kinases and recruits PKCδ to the plasma membrane through tyrosine phosphorylation-dependent association with the C2 domain of PKCδ, which in turn induces a survival signal in an anchorage-independent condition. In this study, we used our cell-free screening system to identify a small compound, glycoconjugated palladium complex (Pd-Oqn), which significantly inhibited the interaction between the C2 domain of PKCδ and phosphorylated CDCP1. Immunoprecipitation assays demonstrated that Pd-Oqn hindered the intercellular interaction of phosphorylated CDCP1 with PKCδ and also suppressed the phosphorylation of PKCδ but not that of ERK or AKT. In addition, Pd-Oqn inhibited the colony formation of gastric adenocarcinoma 44As3 cells in soft agar as well as their invasion. In mouse models, Pd-Oqn markedly reduced the peritoneal dissemination of gastric adenocarcinoma cells and the tumor growth of pancreatic cancer orthotopic xenografts. These results suggest that the novel compound Pd-Oqn reduces tumor metastasis and growth by inhibiting the association between CDCP1 and PKCδ, thus potentially representing a promising candidate among therapeutic reagents targeting protein-protein interaction. [ABSTRACT FROM AUTHOR]

The widespread environmental contaminant, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), is considered one of the most toxic dioxin-like compounds. Although epidemiological studies have shown that TCDD exposure is linked to some neurological and neurophysiological disorders, the underlying mechanism of TCDD-mediated neurotoxicity has remained unclear. Astrocytes are the most abundant cells in the nervous systems, and are recognized as the important mediators of normal brain functions as well as neurological, neurodevelopmental and neurodegenerative brain diseases. In this study, we investigated the role of TCDD in regulating the expression of glutamate transporter GLT-1 in astrocytes. TCDD, at concentrations of 0.1-100 n m, had no significantly harmful effect on the viability of C6 glioma cells. However, the expression of GLT-1 in C6 glioma cells was downregulated in a dose- and time-dependent manner. TCDD also caused activation of protein kinase C (PKC), as TCDD induced translocation of the PKC from the cytoplasm or perinuclear to the membrane. The translocation of PKC was inhibited by one Ca2+ blocker, nifedipine, suggesting that the effects are triggered by the initial elevated intracellular concentration of free Ca2+. Finally, we showed that inhibition of the PKC activity reverses the TCDD-triggered reduction of GLT-1. In summary, our results suggested that TCDD exposure could downregulate the expression of GLT-1 in C6 via Ca2+/PKC pathway. The downregulation of GLT-1 might participate in TCDD-mediated neurotoxicity. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P) is emerging to have hypoxic preconditioning potential in various preclinical studies. The study aims to evaluate the preclinical preconditioning efficacy of exogenously administered S1P against acute hypobaric hypoxia (HH)-induced pathological disturbances. Male Sprague Dawley rats (200 ± 20 g) were preconditioned with 1, 10, and 100 μg/kg body weight (b.w.) S1P (i.v.) for three consecutive days. On the third day, S1P preconditioned animals, along with hypoxia control animals, were exposed to HH equivalent to 7,620 m (280 mm Hg) for 6 h. Postexposure status of cardiac energy production, circulatory vasoactive mediators, pulmonary and cerebral oxidative damage, and inflammation were assessed. HH exposure led to cardiac energy deficit indicated by low ATP levels and pronounced AMPK activation levels, raised circulatory levels of brain natriuretic peptide and endothelin-1 with respect to total nitrate (NOx), redox imbalance, inflammation, and alterations in NOx levels in the pulmonary and cerebral tissues. These pathological precursors have been routinely reported to be coincident with high-altitude diseases. Preconditioning with S1P, especially 1 µg/kg b.w. dose, was seen to reverse the manifestation of these pathological disturbances. The protective efficacy could be attributed, at least in part, to enhanced activity of cardioprotective protein kinase C and activation of small GTPase Rac1, which led to further induction of hypoxia-adaptive molecular mediators: hypoxia-inducible factor (HIF)−1α and Hsp70. This is a first such report, to the best of our knowledge, elucidating the mechanism of exogenous S1P-mediated HIF-1α/Hsp70 induction. Conclusively, systemic preconditioning with 1 μg/kg b.w. S1P in rats protects against acute HH-induced pathological disturbances. © 2016 IUBMB Life 68(5):365-375, 2016 [ABSTRACT FROM AUTHOR]

The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o-proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 ( ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/ PKC-, PLDs-, and epidermal growth factor receptor ( EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions ( OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a vital post-translational modification. This study investigated the effect of phosphorylation on human uridine diphosphate (UDP)-glucuronosyltransferase 1A3 (UGT1A3) activity. Curcumin and calphostin C suppressed the activity and phosphorylation of recombinant UGT1A3 expressed in Sf9 cells. These results indicate that UGT1A3 undergoes phosphorylation, which is required for its catalytic activity. Calphostin C is a highly specific protein kinase C (PKC) inhibitor, so three predicted PKC phosphorylation sites in UGT1A3 were examined. Site-directed mutation analysis at residues 28, 43 and 436 (from serine to glycine) was conducted. Compared with the wild-type, the S43G-mutant showed significantly decreased UGT1A3 catalytic activity. Furthermore, the UGT1A3 activity of wild-type and S43G-mutant was down-regulated by calphostin C, whereas the calphostin C inhibitory effect was much weaker on the S43G-mutant than the wild-type. In conclusion, phosphorylation plays an important role in UGT1A3 activity, and the serine at site 43 in UGT1A3 is most likely a phosphorylation site. [ABSTRACT FROM AUTHOR]

Kujin contains antiallergic compounds that inhibit upregulation of histamine H1 receptor (H1R) and interleukin (IL)-4 gene expression. However, the underlying mechanism remains unknown. We sought to identify a Kujin-derived antiallergic compound and investigate its mechanism of action. The H1R and IL-4 mRNA levels were determined by real-time quantitative RT-PCR. To investigate the effects of maackiain in vivo, toluene-2,4-diisocyanate (TDI)-sensitized rats were used as a nasal hypersensitivity animal model. We identified (-)-maackiain as the responsible component. Synthetic maackiain showed stereoselectivity for the suppression of IL-4 gene expression but not for H1R gene expression, suggesting distinct target proteins for transcriptional signaling. (-)- Maackiain inhibited of PKCd translocation to the Golgi and phosphorylation of Tyr311 on PKCd, which led to the suppression of H1R gene transcription. However, (-)-maackiain did not show any antioxidant activity or inhibition of PKCd enzymatic activity per se. Pretreatment with maackiain alleviated nasal symptoms and suppressed TDI-induced upregulations of H1R and IL-4 gene expressions in TDI-sensitized rats. These data suggest that (-)-maackiain is a novel antiallergic compound that alleviates nasal symptoms in TDI-sensitized allergy model rats through the inhibition of H1R and IL-4 gene expression. The molecular mechanism underlying its suppressive effect for H1R gene expression is mediated by the inhibition of PKCd activation. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, which begins with isolated steatosis and advances to nonalcoholic steatohepatitis (NASH), steatofibrosis, and cirrhosis. The pathways involved in disease progression are not understood. Loss-of-function mutations in Wnt coreceptor LDL receptor-related protein 6 (LRP6) underlie early-onset atherosclerosis, metabolic risk factors, and NAFLD in humans by unknown mechanisms. We generated mice with the human disease-associated LRP6R611C mutation and phenotypically characterized their liver. Homozygote LRP6R611C (LRP6mut/mut) mice exhibited both steatohepatitis and steatofibrosis. These traits were associated with increased activity of the noncanonical Wnt/Ras homolog family member A, Rho-associated protein kinase 2, and PKC- α/µm pathways. Accordingly, there was increased TGF-b1 activity, coupled with enhanced expression of smooth muscle a-actin and vimentin that colocalized with albumin in LRP6mut/mut mouse liver. LRP6 knockdown reprog-ramed HepG2 cells to express both these markers, linking impaired Wnt signaling with hepatocyte trans-differentiation. The causal link between altered Wnt signaling and NASH was established by normalization of the disease pathways and rescue of the liver traits by Wnt3a administration to LRP6mut/mut mice. Thus, this study identifies diverse disease pathways that underlie a spectrum of NASH-related liver diseases and are linked by a single human genetic variant. LRP6 and noncanonical Wnt pathways are important potential therapeutic targets against NASH. [ABSTRACT FROM AUTHOR]

ABSTRACT Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. Microsc. Res. Tech. 78:715-722, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Neuronal excitability depends on the surface concentration of neurotransmitter receptors. Type C gamma-aminobutyric acid receptors (GABAcR) are composed of ρ subunits that are highly expressed in the retina. Molecular mechanisms that guide the surface concentration of this receptor type are largely unknown. Previously, we reported physical interactions of GABACR ρ subunits with protein kinase C-ζ (PKCζ) via adapter proteins of the ZIP protein family, as well as of protein phosphatase 1 (PP1) via PNUTS. Here, we demonstrate that co-expressing ρ1 with ZIP3 and PKCf enhanced basal internalization of GABACR, while receptor internalization was reduced in the presence of PNUTS and PP1. Co-expression of ρ1 with individual binding partners showed no alterations, except for PP1. Heterooligomeric GABACR composed of ρ1 and ρ2 subunits had a significant higher endocytosis rate than ρ1 containing homooligomeric receptors. Mutant constructs lacking binding sites for protein interactions ensured the specificity of our data. Finally, substitution of serine and threonine residues with alanines indicated that GABACR internalization depends on serine/threonine kinases and phosphatases, but not on tyrosine phosphorylation. We conclude that GABACR internalization is reciprocally regulated by PKCf and PP1 that are anchored to the receptor via ZIP3 or PNUTS respectively. [ABSTRACT FROM AUTHOR]

Oxaliplatin is a key drug commonly used in colorectal cancer treatment. Despite high clinical efficacy, its therapeutic application is limited by common, dose-limiting occurrence of neuropathy. As usual symptomatic neuropathy treatments fail to improve the patients' condition, there is an urgent need to advance our understanding of the pathogenesis of neuropathy to propose effective therapy and ensure adequate pain management. Oxaliplatin-induced neuropathy was recently reported to be associated with protein kinase C (PKC) activation. It is unclear, however, whether PKC inhibition can prevent neuropathy. In our current studies, we found that a PKC inhibitor, tamoxifen, inhibited oxaliplatin-induced neuropathy via the PKC/extracellular signal-regulated kinase (ERK)/c-Fos pathway in lumbar spinal cords (lumbar segments 4-6). Additionally, tamoxifen was shown to act in synergy with oxaliplatin to inhibit growth in tumor cells-implanted mice. Moreover, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, PD0325901, suppressed oxaliplatin-induced neuropathy and enhanced oxaliplatin efficacy. Our results indicate that oxaliplatin-induced neuropathy is associated with PKC/ERK/c-Fos pathway in lumbar spinal cord. Additionally, we demonstrate that disruption of this pathway by PKC and MEK inhibitors suppresses oxaliplatin-induced neuropathy, thereby suggesting that PKC and MEK inhibitors may be therapeutically useful in preventing oxaliplatin-induced neuropathy and could aid in combination antitumor pharmacotherapy. [ABSTRACT FROM AUTHOR]

In clinic, we examined the expression of protein kinase C ( PKC)-α and Dicer in the samples of bladder cancer patients, and found that the two proteins have a line correlation. Our study confirmed this correlation existing by clearing the decreasing expression of Dicer after the PKC-α knockdown. Treatment of bladder cancer cell lines (T24, 5637) with the PKC-α or Dicer knockdown and the PKC inhibitors (Calphostin C and Gö 6976) can promote the apoptosis. Inhibition of PKC can increase the activities of caspase-3 and PARP, however, decrease the expression of Dicer. And knockdown of the PKC-α or Dicer can also activate the caspase-3 or the PARP. Considering the reduction of PKC-α can induce the Dicer down-regulation, we make the conclusion that the reduction of PKC-α can promote the apoptosis via the down-regulation of Dicer in bladder cancer. [ABSTRACT FROM AUTHOR]

Large tumors exhibit high interstitial pressure heightened by growth against the constraining stroma. Such pressures could stimulate tumor proliferation via a mechanosensitive ion channel. We studied the effects of 0–80 mmHg increased extracellular pressure for 24 h on proliferation of SW620, Caco-2, and CT-26 colon; MCF-7 breast; and MLL and PC3 prostate cancer cells, and delineated its mechanism in SW620 cells with specific inhibitors and siRNA. Finally, we compared NF-kB, phospho-IkB and cyclin D1 immunoreactivity in the high pressure centers and low pressure peripheries of human tumors. Pressure-stimulated proliferation in all cells. Pressure-driven SW620 proliferation required calcium influx via the T-type Ca 2+ channel Cav3.3, which stimulated PKC-β to invoke the IKK-IkB–NF–kB pathway to increase proliferation and S-phase fraction. The mitotic index and immunoreactivity of NF-kB, phospho-IkB, and cyclin D1 in the center of 28 large human colon, lung, and head and neck tumors exceeded that in tumor peripheries. Extracellular pressure increases [Ca 2+ ]i via Cav3.3, driving a PKC-β- IKK- IkB–NF–kB pathway that stimulates cancer cell proliferation. Rapid proliferation in large stiff tumors may increase intratumoral pressure, activating this pathway to stimulate further proliferation in a feedback cycle that potentiates tumor growth. Targeting this pathway may inhibit proliferation in large unresectable tumors. [ABSTRACT FROM AUTHOR]

Background and Purpose: The majority of studies examining desensitization of the μ-opioid receptor (MOR) have examined those located at cell bodies. However, MORs are extensively expressed at nerve terminals throughout the mammalian nervous system. This study is designed to investigate agonist-induced MOR desensitization at nerve terminals in the mouse ventral tegmental area (VTA).Experimental Approach: MOR function was measured in mature mouse brain slices containing the VTA using whole-cell patch-clamp electrophysiology. Presynaptic MOR function was isolated from postsynaptic function and the functional selectivity, time-dependence and mechanisms of agonist-induced MOR desensitization were examined.Key Results: MORs located at GABAergic nerve terminals in the VTA were completely resistant to rapid desensitization induced by the high-efficacy agonists DAMGO and Met-enkephalin. MORs located postsynaptically on GABAergic cell bodies readily underwent rapid desensitization in response to DAMGO. However, after prolonged (>7 h) treatment with Met-enkephalin, profound homologous MOR desensitization was observed. Morphine could induce rapid MOR desensitization at nerve terminals when PKC was activated.Conclusions and Implications: Agonist-induced MOR desensitization in GABAergic neurons in the VTA is compartment-selective as well as agonist-selective. When MORs are located at cell bodies, higher-efficacy agonists induce greater levels of rapid desensitization than lower-efficacy agonists. However, the converse is true at nerve terminals where agonists that induce MOR desensitization via PKC are capable of rapid agonist-induced desensitization while higher-efficacy agonists are not. MOR desensitization induced by higher-efficacy agonists at nerve terminals only takes place after prolonged receptor activation.Linked Articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. [ABSTRACT FROM AUTHOR]

Unlabelled: The efficiency of μ-opioid receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific μ-opioid receptor antibodies, which have proved to be excellent tools for monitoring the spatial and temporal dynamics of receptor phosphorylation and dephosphorylation. Agonist-induced phosphorylation of μ-opioid receptors occurs at a conserved 10 residue sequence (370) TREHPSTANT(379) in the receptor's carboxyl-terminal cytoplasmic tail. Diverse opioids induce receptor phosphorylation at S375, present in the middle of this sequence, but only high-efficacy opioids have the ability to drive higher order phosphorylation on flanking residues (T370, T376 and T379). S375 is the initiating residue in a hierarchical phosphorylation cascade. In contrast, agonist-independent heterologous μ-opioid receptor phosphorylation occurs primarily at T370. The combination of phosphosite-specific antibodies and siRNA knockdown screening also facilitated the identification of relevant kinases and phosphatases. In fact, morphine induces a selective S375 phosphorylation that is predominantly catalysed by GPCR kinase 5 (GRK5), whereas multisite phosphorylation induced by high-efficacy opioids specifically requires GRK2/3. By contrast, T370 phosphorylation stimulated by phorbol esters or heterologous activation of Gq -coupled receptors is mediated by PKCα. Rapid μ-opioid receptor dephosphorylation occurs at or near the plasma membrane and is catalysed by protein phosphatase 1γ (PP1γ). These findings suggest that there are distinct phosphorylation motifs for homologous and heterologous regulation of μ-opioid receptor phosphorylation. However, it remains to be seen to what extent different μ-opioid receptor phosphorylation patterns contribute to the development of tolerance and dependence in vivo.Linked Articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. [ABSTRACT FROM AUTHOR]

Background and Purpose This study examined the role of agents known to activate PKC on morphine-induced desensitization of μ-opioid receptors ( MOP receptors) in brain slices containing locus coeruleus neurons. Experimental Approach Intracellular recordings were obtained from rat locus coeruleus neurons. Two measurements were used to characterize desensitization, the decline in hyperpolarization induced by application of a saturating concentration of agonist (acute desensitization) and the decrease in hyperpolarization induced by a subsaturating concentration of [ Met]5enkephalin ( ME) following washout of the saturating concentration (sustained desensitization). Internalization of MOP receptors was studied in brain slices prepared from transgenic mice expressing Flag- MOP receptors. The subcellular distribution of activated PKC was examined using a novel fluorescent sensor of PKC in HEK293 cells. Key Results The phorbol esters (PMA and PDBu) and muscarine increased acute desensitization induced by a saturating concentration of morphine and ME. These effects were not sensitive to staurosporine. Staurosporine did not block the decline in hyperpolarization induced by muscarine. PDBu and muscarine did not affect sustained desensitization induced by ME nor did phorbol esters or muscarine change the trafficking of MOP receptors induced by morphine or ME. The distribution of activated PKC measured in HEK293 cells differed depending on which phorbol ester was applied. Conclusions and Implications This study demonstrates a distinct difference in two measurements that are often used to evaluate desensitization. The measure of decline correlated well with the reduction in peak amplitudes caused by PKC activators implicating the modification of other factors rather than MOP receptors. Linked Articles This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit [ABSTRACT FROM AUTHOR]

The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H 2 O 2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H 2 O 2 -induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells. [ABSTRACT FROM AUTHOR]

Pancreatic cancer is one of the most lethal human cancers, and radiotherapy is often implemented for locally advanced pancreatic ductal adenocarcinoma. Tumor cell repopulation is a major challenge in treating cancers after radiotherapy. In order to address the problem of tumor repopulation, our previous studies have demonstrated that dying cells stimulate the proliferation of living tumor cells after radiotherapy. In particular, dying cells undergoing apoptosis also activate survival or proliferation signals and release growth factors to surrounding living cells. In the present study, we used an in vitro model to examine the possible mechanisms for dying cell stimulated tumor repopulation in pancreatic cancer. In this model, a small number of living, luciferase-labeled pancreatic cancer cells (reporter) were seeded onto a layer of a much larger number of irradiated, unlabeled pancreatic cancer cells and the growth of the living cells was measured over time as a gage of tumor repopulation. Our results indicate that irradiated, dying Panc1 feeder cells significantly stimulated the proliferation of living Panc1 reporter cells. Importantly, we identified that the percentage of apoptotic cells and the cleavage of caspases 3 and 7 and protein kinase Cδ (PKCδ) were increased in irradiated Panc1 cells. We presumed that caspases 3 and 7 and PKCδ as integral mediators in the process of dying pancreatic cancer cell stimulation of living tumor cell growth. In order to demonstrate the importance of caspases 3, 7 and PKCδ, we introduced dominant-negative mutants of caspase 3 (DN_C3), caspase 7 (DN_C7), or PKCδ (DN_PKCδ) into Panc1 cells using lentiviral vectors. The stably transduced Panc1 cells were irradiated and used as feeders and we found a significant decrease in the growth of living Panc1 reporter cells when compared with irradiated wild-type Panc1 cells as feeders. Moreover, the role of PKCδ in the growth stimulation of living tumor cells was further confirmed using a pan PKC inhibitor GF109203x and a specific PKCδ inhibitor, rottlerin. Additionally, we found significantly increased phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK1/2) in the irradiated Panc1 cells. Mechanistically, PKCδ cleavage was attenuated in both DN_C3 and DN_C7 transduced Panc1 cells, and both Akt and p38 MAPK phosphorylation were attenuated in DN_PKCδ transduced Panc1 cells following radiation. Thus, this report suggests a novel finding that cellular signaling caspase 3/7-PKCδ-Akt/p38 MAPK is crucial to the repopulation in Panc1 cells after radiotherapy. [ABSTRACT FROM AUTHOR]