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Dentale Follikelzellen (DFCs, engl. Dental follicle cells) sind die endogenen Stamm-/Vorläuferzellen des Zahnhalteapparats und können in alveoläre Osteoblasten, Zementoblasten und Fibroblasten des parodontalen Ligaments differenzieren. DFCs besitzen großes Potential für regenerative Therapien von (dentalem) Knochengewebe, wobei zunächst eine genaue Kenntnis der molekularen Mechanismen während der osteogenen Differenzierung, welche sich in vitro durch BMP2 (engl. Bone morphogenetic protein 2) oder Dexamethason induzieren lässt, erforderlich ist. Nachdem Vorarbeiten zeigten, dass klassische Isoformen der Proteinkinase C (PKC) während der Osteogenese von DFCs herunterreguliert werden und diese hemmen, sollten in der vorliegenden Arbeit die zugrundeliegenden Mechanismen evaluiert werden. Hierfür wurde die Regulierung potentieller Zielproteine und Signalwege nach osteogener Induktion und Hemmung klassischer PKCs untersucht sowie deren Einfluss auf osteogene Marker evaluiert. Dabei wurde festgestellt, dass klassische PKCs die Kinase Akt regulieren und stromabwärts durch Phosphorylierung von GSK3β (Glykogensynthase-Kinase 3β) die Aktivität des Proteins β-Catenin beeinflussen, welches eine wichtige Rolle für die Induktion der Osteogenese spielt. Während Akt zwar die Expression von aktivem β-Catenin unterstützte, führte eine Überaktivierung der Kinase jedoch zur Inhibition der Differenzierung und des Signalwegs stromabwärts von BMP2. Darüber hinaus konnte eine Regulierung des NF-κB (engl. Nuclear factor kappa B)-Signalwegs durch klassische PKCs und Akt nachgewiesen werden, wobei NF-κB die Proliferation und osteogene Differenzierung von DFCs unterdrückte. Dahingegen konnte keine direkte Beteiligung von klassischen PKCs oder Akt an der BMP2-induzierten Aktivierung der Proteinkinase A (PKA), welche die Osteogenese unterstützt, gefunden werden. Stattdessen zeigten die Versuche, dass die PKA-Aktivierung von der intrazellulären Expression des Proteins PTHrP (engl. Parathyroid hormone-related protein) abhängt, und dass PKA den Signalweg stromabwärts von BMP2 durch eine negative Rückkopplung hemmt. Des Weiteren konnte eine Induktion des mitochondrialen Energiemetabolismus und des oxidativen Stresses während der osteogenen Differenzierung und nach Inhibition klassischer PKCs festgestellt werden, während eine Hemmung des mitochondrialen Stoffwechsels die Mineralisierung unterdrückte. Eine Evaluierung von Proteinen, welche oxidativen Stress neutralisieren können, zeigte eine Stimulierung der Catalase-Expression und eine Herunterregulierung der Expression von GPX1 (Glutathionperoxidase 1) während der Differenzierung und nach Inhibition klassischer PKCs. Überdies führte eine Hemmung der Catalase zu einer verringerten Mineralisierung. Stromaufwärts von PKC wurde PTHrP für die Aktivität der Kinase benötigt, während das Protein WNT5A (engl. Wnt family member 5A) diese unterdrückte. Die Ergebnisse legten dar, dass klassische Isoformen von PKC die osteogene Differenzierung von DFCs über verschiedene Mechanismen hemmen, wobei insbesondere biologische Prozesse reguliert werden, die in späteren Differenzierungsphasen wichtig sind., Dental follicle cells (DFCs) are the genuine stem/precursor cells of the periodontium and capable of differentiating into alveolar osteoblasts, cementoblasts and periodontal ligament fibroblasts. DFCs possess great potential for regenerative therapies of (dental) bone tissue, but their usage first requires a profound knowledge of the molecular mechanisms during the osteogenic differentiation, which can be induced in vitro by BMP2 (Bone morphogenetic protein 2) or dexamethasone. After previous experiments showed that classical isoforms of PKC (Protein kinase C) are downregulated during osteogenesis of DFCs and inhibit the differentiation, this thesis aimed to evaluate the underlying mechanisms. For this, regulation of potential target proteins and signaling pathways was analyzed after osteogenic induction and inhibition of classical PKCs, and their impact on osteogenic markers was investigated. The results showed that classical PKCs regulate the kinase Akt, which in turn phosphorylates GSK3β (Glycogen synthase kinase 3β) and consequently affects the stability of β-catenin, which plays an important role for the induction of osteogenesis. While Akt supported the expression of active β-catenin, overactivation of the kinase, however, inhibited the differentiation and the signaling pathway downstream of BMP2. Furthermore, classical PKCs and Akt regulated the NF-κB (Nuclear factor kappa B) signaling pathway, and NF-κB was shown to suppress proliferation and osteogenic differentiation of DFCs. By contrast, classical PKCs and Akt were not directly involved in the BMP2-induced activation of protein kinase A (PKA), which is known to support osteogenesis. Instead, the experiments showed that activation of PKA depends on the intracellular expression of the protein PTHrP (Parathyroid hormone-related protein) and that PKA inhibits the signaling pathway downstream of BMP2 via negative feedback. Moreover, mitochondrial energy metabolism and oxidative stress were induced during the osteogenic differentiation as well as after inhibition of classical PKCs, and inhibition of the mitochondrial metabolism suppressed mineralization. Evaluation of proteins which are capable of neutralizing oxidative stress revealed that expression of catalase was upregulated and expression of GPX1 (Glutathione peroxidase 1) was inhibited during differentiation and after inhibition of classical PKCs. Besides, inhibition of catalase attenuated mineralization. Upstream of PKC, PTHrP was required for kinase activity, while WNT5A (Wnt family member 5A) suppressed it. The results showed that classical isoforms of PKC inhibit the osteogenic differentiation of DFCs via different mechanisms, which especially involve biological processes important for later differentiation periods.

In comparing two human lung cancer cells, we previously found lower levels of acetylcholinesterase (AChE) and intact amyloid-β40/42 (Aβ), and higher levels of mature brain-derived neurotrophic factor (mBDNF) in the media of H1299 cells as compared to A549 cell media. In this study, we hypothesized that the levels of soluble amyloid precursor protein α (sAPPα) are regulated by AChE and mBDNF in A549 and H1299 cell media. The levels of sAPPα were higher in the media of H1299 cells. Knockdown of AChE led to increased sAPPα and mBDNF levels and correlated with decreased levels of intact Aβ40/42 in A549 cell media. AChE and mBDNF had opposite effects on the levels of Aβ and sAPPα and were found to operate through a mechanism involving α-secretase activity. Treatment with AChE decreased sAPPα levels and simultaneously increased the levels of intact Aβ40/42 suggesting a role of the protein in shifting APP processing away from the non-amyloidogenic pathway and toward the amyloidogenic pathway, whereas treatment with mBDNF led to opposite effects on those levels. We also show that the levels of sAPPα are regulated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)1/2, phosphoinositide 3 Kinase (PI3K), but not by protein kinase A (PKA).

Bastian Welz,* Rolf Bikker,* Leonie Hoffmeister, Mareike Diekmann, Martin Christmann, Korbinian Brand,* René Huber* Institute of Clinical Chemistry, Hannover Medical School, Hannover, 30625, Germany*These authors contributed equally to this workCorrespondence: Korbinian BrandInstitute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg Str. 1, Hannover, 30625, GermanyTel +49 511 532 6614Fax +49 511 532 8614Email brand.korbinian@mh-hannover.deBackground: Termination of TNF-induced signaling plays a key role in the resolution of inflammation with dysregulations leading to severe pathophysiological conditions (sepsis, chronic inflammatory disease, cancer). Since a recent phospho-proteome analysis in human monocytes suggested GSK3 as a relevant kinase during signal termination, we aimed at further elucidating its role in this context.Materials and Methods: For the analyses, THP-1 monocytic cells and primary human monocytes were used. Staurosporine (Stauro) was applied to activate GSK3 by inhibiting kinases that mediate inhibitory GSK3α/β-Ser21/9 phosphorylation (eg, PKC). For GSK3 inhibition, Kenpaulone (Ken) was used. GSK3- and PKC-siRNAs were applied for knockdown experiments. Protein expression and phosphorylation were assessed by Western blot or ELISA and mRNA expression by qPCR. NF-κB activation was addressed using reporter gene assays.Results: Constitutive GSK3β and PKCβ expression and GSK3α/β-Ser21/9 and PKCα/βII-Thr638/641 phosphorylation were not altered during TNF long-term incubation. Stauro-induced GSK3 activation (demonstrated by Bcl3 reduction) prevented termination of TNF-induced signaling as reflected by strongly elevated IL-8 expression (used as an indicator) following TNF long-term incubation. A similar increase was observed in TNF short-term-exposed cells, and this effect was inhibited by Ken. PKCα/β-knockdown modestly increased, whereas GSK3α/β-knockdown inhibited TNF-induced IL-8 expression. TNF-dependent activation of two NF-κB-dependent indicator plasmids was enhanced by Stauro, demonstrating transcriptional effects. A TNF-induced increase in p65-Ser536 phosphorylation was further enhanced by Stauro, whereas IκBα proteolysis and IKKα/β-Ser176/180 phosphorylation were not affected. Moreover, PKCβ-knockdown reduced levels of Bcl3. A20 and IκBα mRNA, both coding for signaling inhibitors, were dramatically less affected under our conditions when compared to IL-8, suggesting differential transcriptional effects.Conclusion: Our results suggest that GSK3 activation is involved in preventing the termination of TNF-induced signaling. Our data demonstrate that activation of GSK3 – either pathophysiologically or pharmacologically induced – may destroy the finely balanced condition necessary for the termination of inflammation-associated signaling.Keywords: TNF, GSK3, PKC, staurosporine, IL-8, NF-κB, termination of TNF-induced signaling, termination of inflammation

In addition to its function as an intravascular lipid transporter, LDL also triggers signal transduction in endothelial cells (ECs), which, among other things, trigger immunomodulatory cascades, e.g., IL-6 upregulation. However, the molecular mechanisms of how these LDL-triggered immunological responses in ECs are realized are not fully understood. Since promyelocytic leukemia protein (PML) plays a role in promoting inflammatory processes, we examined the relationship between LDL, PML, and IL-6 in human ECs (HUVECs and EA.hy926 cells). RT-qPCR, immunoblotting, and immunofluorescence analyses showed that LDL but not HDL induced higher PML expression and higher numbers of PML-nuclear bodies (PML-NBs). Transfection of the ECs with a PML gene-encoding vector or PML-specific siRNAs demonstrated PML-regulated IL-6 and IL-8 expression and secretion after LDL exposure. Moreover, incubation with the PKC inhibitor sc-3088 or the PKC activator PMA showed that LDL-induced PKC activity leads to the upregulation of PML mRNA and PML protein. In summary, our experimental data suggest that high LDL concentrations trigger PKC activity in ECs to upregulate PML expression, which then increases production and secretion of IL-6 and IL-8. This molecular cascade represents a novel cellular signaling pathway with immunomodulatory effects in ECs in response to LDL exposure.

Graphical abstract, Highlights • Epinephrine induced MI with renal complication through Nrf2/NF-κB imbalance and PKC/AT1R. • Ginseng abolished ECG changes induced by epinephrine and stimulated Nrf2. • Ginseng reduced upregulation of PKC, NF-κB, and AT1R induced by epinephrine. • Ginseng inhibited iNOS and corrected renal disorder in epinephrine model of MI., The expression of angiotensin II type 1 receptor (AT1 receptor)/protein kinase C (PKC) in heart tissues has a vital role in myocardial infarction (MI). The current work aimed to clarify the renal complication enhanced by MI following epinephrine injection via AT1 receptor/ PKC expression; in addition, the impact of ginseng extract on epinephrine-induced MI and its renal complication was assessed. Adult male albino Wistar rats were pretreated orally with ginseng extract (200 & 400 mg/kg/day) for 14 days, then two successive doses of epinephrine injection (100 mg/kg, i.p.). Epinephrine evoked electrocardiographic (ECG) and renal changes accompanied with a significant increase in heart and kidney contents of malodialdehyde (MDA), nitric oxide (NO), protein kinase C (PKC), heart contents of nuclear factor-kabba B (NF-κB) and angiotensin 1receptor (AT1R), as well as a decrease in heart and kidney reduced glutathione (GSH) and nuclear factor-erythroid-related factor 2 (Nrf2) contents. In histopathological investigations epinephrine exhibited deleterious heart changes in the form of acute MI with the presence of necrosis of cardiomyocytes with iNOS expression and produced glomerulus and renal tubules degeneration. Pretreatment of rats with ginseng extract in both doses significantly corrected epinephrine-induced heart and renal changes. The current work revealed that epinephrine-induced MI associated with aggravated renal complication and ginseng extract has cardio and reno protective role against this as it reduces infarct size, preserves cardiac and renal tissues and functions through activating Nrf2 and down-regulating NF-κB, PKC, AT1R and iNOS.

Although the previous research confirmed that triclosan (TCS) induced an estrogen effect by acting on a novel G-protein coupled estrogen-membrane receptor (GPER), the underlying mechanisms by which downstream pathways induce neurotoxicity remain unclear after TCS activation of GPER. By employing a series of techniques (Illumina miRNA-seq, RT-qPCR, and artificial intervention of miRNA expression), we screened out four important miRNAs, whose target genes were directly/indirectly involved in neurodevelopment and neurobehavior. Especially, the miR-144 up-regulation caused vascular malformation and severely affected hair-cell development and lateral-line-neuromast formation, thereby causing abnormal motor behavior. After microinjecting 1-2-cell embryos, the similar phenotypic malformations as those induced by TCS were observed, including aberrant neuromast, cuticular-plate development and motor behavior. By KEGG pathway enrichment analysis, these target genes were demonstrated to be mainly related to the PKC/MAPK signaling pathway. When a PKC inhibitor was used to suppress the PKC/MAPK pathway, a substantial alleviation of TCS-induced neurotoxicity was observed. Therefore, TCS acts on GPER to activate the downstream PKC/MAPK signaling pathway, further up-regulating miR-144 expression and causing abnormal modulation of these nerve-related genes to trigger neurodevelopmental toxicity. These findings unravel the molecular mechanisms of TCS-induced neurodegenerative diseases, and offer theoretical guidance for TCS-pollution early warning and management.

While irradiation with red LED light has been reported to modulate sperm function in different mammalian species, the mechanisms underlying their response are poorly understood. This work sought to provide new insights into whether this effect relies on a direct action upon mitochondrial electron chain and/or on PKC-linked mechanisms such as those related to opsins. For this purpose, pig semen was light-stimulated for 1 min, 5 min or 10 min in the presence/absence of antimycin A, an inhibitor of the mitochondrial electron chain, or PKC 20-28® (PKCi), a PKC inhibitor. Antimycin A completely blocked the effects of light at all the performed irradiation patterns. This effect was linked to a complete immobility of sperm, which was accompanied with a significant (P

Leucine, nutrient signal and substrate for the branched chain aminotransferase (BCAT) activates the mechanistic target of rapamycin (mTORC1) and regulates autophagic flux, mechanisms implicated in the pathogenesis of neurodegenerative conditions such as Alzheimer's disease (AD). BCAT is upregulated in AD, where a moonlighting role, imparted through its redox-active CXXC motif, has been suggested. Here we demonstrate that the redox state of BCAT signals differential phosphorylation by protein kinase C (PKC) regulating the trafficking of cellular pools of BCAT. We show inter-dependence of BCAT expression and proteins associated with the P13K/Akt/mTORC1 and autophagy signalling pathways. In response to insulin or an increase in ROS, BCATc is trafficked to the membrane and docks via palmitoylation, which is associated with BCATc-induced autophagy through PKC phosphorylation. In response to increased levels of BCATc, as observed in AD, amyloid β (Aβ) levels accumulate due to a shift in autophagic flux. This effect was diminished when incubated with leucine, indicating that dietary levels of amino acids show promise in regulating Aβ load. Together these findings show that increased BCATc expression, reported in human AD brain, will affect autophagy and Aβ load through the interdependence of its redox-regulated phosphorylation offering a novel target to address AD pathology.

Protein kinase C (PKC) is a protein kinase with important cellular functions. PKC-δ, a member of the novel PKC subfamily, has been well-documented over the years. Activation of PKC-δ plays an important regulatory role in myocardial ischemia/reperfusion (IRI) injury and myocardial fibrosis, and its activity and expression levels can regulate pathological cardiovascular diseases such as atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. This article aims to review the structure and function of PKC-δ, summarize the current research regarding its activation mechanism and its role in cardiovascular disease, and provide novel insight into further research on the role of PKC-δ in cardiovascular diseases.

BackgroundG protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH.MethodsHere we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein–protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage.ResultsWe identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation.ConclusionOur results show a stimulated shift of PP2Ac from PI3K to AKT termed “PP2A switch” that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation.

Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous disease characterized by persistent airflow limitation but still lacking effective treatments. Perilla frutescens (L.) Britt., an important traditional medicinal plant with excellent antioxidant and anti-inflammatory properties, is widely used for the treatment of respiratory disease in China. However, its protective activity and mechanism against COPD airway inflammation have not been fully studied. Here, the anti-inflammatory effects of the PLE were investigated, and its underlying mechanisms were then elucidated. The presented results suggested a notable effect of the PLE on airway inflammation of COPD, by significantly ameliorating inflammatory cell infiltration in lung tissue, lessening leukocytes (lymphocytes, neutrophils, and macrophages) and inflammatory mediators (interleukin 4 (IL-4), IL-6, IL-17A, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α)) in the bronchoalveolar lavage fluid (BALF) of cigarette smoke (CS)/lipopolysaccharide (LPS)-induced COPD mice in vivo and inhibiting the production of inflammatory factors (nitric oxide (NO), IL-6, and TNF-α) and intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells in vitro. For further extent, PLE treatment significantly suppressed the expression and phosphorylation of TLR4, Syk, PKC, and NF-κB p65 in vivo and their mRNA in vitro. Subsequently, by co-treating with their inhibitors in vitro, its potential mechanism via TLR4/Syk/PKC/NF-κB p65 signals was disclosed. In summary, the obtained results indicated a noteworthy effective activity of the PLE on COPD inflammation, and partly, the TLR4/Syk/PKC/NF-κB p65 axis might be the potential mechanism.

Major depression disorder (MDD) is a severe mental alteration with a multifactorial origin, and chronic stress is one of the most relevant environmental risk factors associated with MDD. Although there exist some therapeutical options, 30% of patients are still resistant to any type of treatment. GSK3β inhibitors are considered very promising therapeutic tools to counteract stress-related affectations. However, they are often associated with excessive off-target effects and undesired secondary alterations. Meridianins are alkaloids with an indole framework linked to an aminopyrimidine ring from Antarctic marine ascidians. Meridianins could overcome several of the aforementioned limitations since we previously demonstrated that they can inhibit GSK3β activity without the associated neurotoxic or off-target effects in rodents. Here, we show that meridianins delivered into the lateral ventricle inhibited GSK3β in several brain regions involved with stress-related symptoms. We also observed changes in major signaling pathways in the prefrontal cortex (Akt and PKA) and hippocampus (PKC and GluR1). Moreover, meridianins increased synaptic activity, specifically in the CA1 but not in the CA3 or other hippocampal subfields. Finally, we chronically treated the mice subjected to an unpredictable mild chronic stress (CUMS) paradigm with meridianins. Our results showed improvements produced by meridianins in behavioral alterations provoked by CUMS. In conclusion, meridianins could be of therapeutic interest to patients with stress-related disorders such as MDD.

Diabetic rat embryos have increased cortical neurogenesis and neuron maturation, and their offspring presented altered neuron polarity, lamination, and diminished neuron excitability. The FOXP2 overexpression results in higher cortical neurogenesis by increasing the transition of radial glia to the intermediate progenitor. Similarly, histamine through H1-receptor activation increases cortical neuron differentiation. Indeed, blocking the H1-receptor by the systemic administration of chlorpheniramine to diabetic pregnant rats prevents increased neurogenesis. Here, we explore the relationship between the H1-receptor and FOXP2 on embryo neurogenesis from diabetic dams. Through qRT-PCR, Western blot, immunohistofluorescence, and flow cytometry, we showed an increased FOXP2 expression and nuclear localization, a reduced Nestin expression and -positive cells number, and a higher PKCα expression in the cortical neuroepithelium of fourteen-day-old embryos from diabetic rats. Interestingly, this scenario was prevented by the chlorpheniramine systemic administration to diabetic pregnant rats at embryo day twelve. These data, together with the bioinformatic analysis, suggest that higher H1-receptor activity in embryos under high glucose increases FOXP2 nuclear translocation, presumably through PKCα phosphorylation, impairing the transition of radial glia to intermediate progenitor and increasing neuron differentiation in embryos of diabetic rats.

Inhibiting the excessive release of glutamate in the brain is emerging as a promising therapeutic option and is efficient for treating neurodegenerative disorders. The aim of this study is to investigate the effect and mechanism of plantainoside D (PD), a phenylenthanoid glycoside isolated from Plantago asiatica L., on glutamate release in rat cerebral cortical nerve terminals (synaptosomes). We observed that PD inhibited the potassium channel blocker 4-aminopyridine (4-AP)-evoked release of glutamate and elevated concentration of cytosolic Ca2+. Using bafilomycin A1 to block glutamate uptake into synaptic vesicles and EDTA to chelate extracellular Ca2+, the inhibitory effect of PD on 4-AP-evoked glutamate release was prevented. In contrast, the action of PD on the 4-AP-evoked release of glutamate in the presence of dl-TBOA, a potent nontransportable inhibitor of glutamate transporters, was unaffected. PD does not alter the 4-AP-mediated depolarization of the synaptosomal membrane potential, suggesting that the inhibitory effect of PD on glutamate release is associated with voltage-dependent Ca2+ channels (VDCCs) but not the modulation of plasma membrane potential. Pretreatment with the Ca2+ channel blocker (N-type) ω-conotoxin GVIA abolished the inhibitory effect of PD on the evoked glutamate release, as did pretreatment with the protein kinase C inhibitor GF109203x. However, the PD-mediated inhibition of glutamate release was eliminated by applying the mitochondrial Na+/Ca2+ exchanger inhibitor CGP37157 or dantrolene, which inhibits Ca2+ release through ryanodine receptor channels. These data suggest that PD mediates the inhibition of evoked glutamate release from synaptosomes primarily by reducing the influx of Ca2+ through N-type Ca2+ channels, subsequently reducing the protein kinase C cascade.

Perinatal malnutrition affects vascular functions, and calcium is important in vascular regulations. It is unknown whether and how perinatal maternal high-fat diets (MHF)-mediated vascular dysfunction occurs via the angiotensin-PKC-L-type-calcium-channels (LTCC) axis. This study determined angiotensin II (AII) roles in the PKC-LTCC axis in controlling calcium influx in the arteries of offspring after perinatal MHF. Mesenteric arteries (MA) and smooth muscle cells (SMCs) from 5-month-old offspring rats were studied using physiological, ion channel, molecular, and epigenetic analysis. Pressor responses to AII were significantly increased in the free-moving MHF offspring rats. In cell experiments, MA-SMC proliferation was enhanced, and associated with thicker vascular wall in the obese offspring. Imaging analysis showed increase of fluorescence Ca2+ intensity in the SMCs of the MHF group. Angiotensin II receptor (AT1R)-mediated PKC-LTCC axis in vasoconstrictions was altered by perinatal MHF via reduced DNA methylation at specific CpG sites of Agtr1a and Prkcb gene promoters at the transcription level. Accordingly, mRNA and protein expression of AT1R and PKCβ in the offspring MA were increased, contributing to enhanced Ca2+ currents and vascular tone. The results showed that DNA methylation resulted in perinatal MHF-induced vascular disorders via altered AT1-PKC-LTCC pathway in resistance arteries of the offspring, providing new insights into the pathogenesis and early prevention/treatments for hypertension in developmental origins.

The dorsal motor nucleus of the vagus (DMV) contains preganglionic motor neurons important for interpreting sensory input from the periphery, integrating that information, and coding the appropriate parasympathetic (vagal) output to target organs. Despite the critical role of hormonal regulation of vagal motor output, few studies examine the role of neurosteroids in the regulation of the DMV. Of the few examinations, no studies have investigated the potential impact of allopregnanolone (Allo), a neuroactive progesterone-derivative, in the regulation of neurotransmission on the DMV. Since DMV neuronal function is tightly regulated by GABAA receptor activity and Allo is an endogenous GABAA receptor ligand, the present study used in vitro whole cell patch clamp to investigate whether Allo alters GABAergic neurotransmission to DMV neurons. Although Allo did not influence GABAergic neurotransmission during initial application (5–20 min), a TTX-insensitive prolongment of decay time and increase in frequency of GABAergic currents was established after Allo was removed from the bath for at least 30 min (LtAllo). Inhibition of protein kinase C (PKC) abolished these effects, suggesting that PKC is largely required to mediate Allo-induced inhibition of the DMV. Using mice that lack the δ-subunit of the GABAA receptor, we further confirmed that PKC-dependent activity of LtAllo required this subunit. Allo also potentiated GABAA receptor activity after a repeated application of δ-subunit agonist, suggesting that the presence of Allo encodes stronger δ-subunit-mediated inhibition over time. Using current clamp recording, we demonstrated that LtAllo-induced inhibition is sufficient to decrease action potential firing and excitability within DMV neurons. We conclude that the effects of LtAllo on GABAergic inhibition are dependent on δ-subunit and PKC activation. Taken together, DMV neurons can undergo long lasting Allo-dependent GABAA receptor plasticity.

We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.

The internalization of G protein–coupled receptors (GPCRs) can be regulated by PKC. However, most tools available to study the contribution of PKC isozymes have considerable limitations, including a lack of selectivity. In this study, we generated and characterized human embryonic kidney 293A (HEK293A) cell lines devoid of conventional or novel PKC isozymes (ΔcPKC and ΔnPKC) and employ these to investigate the contribution of PKC isozymes in the internalization of the metabotropic glutamate receptor 5 (mGlu5). Direct activation of PKC and mutation of rat mGlu5a Ser901, a PKC-dependent phosphorylation site in the receptor C-tail, both showed that PKC isozymes facilitate approximately 40% of the receptor internalization. Nonetheless, we determined that mGlu5a internalization was not altered upon the loss of cPKCs or nPKCs. This indicates that isozymes from both classes are involved, compensate for the absence of the other class, and thus fulfill dispensable functions. Additionally, using the Gαq/11 inhibitor YM-254890, GPCR kinase 2 and 3 (GRK2 and GRK3) KO cells, and a receptor containing a mutated putative adaptor protein complex 2 (AP-2) interaction motif, we demonstrate that internalization of rat mGlu5a is mediated by Gαq/11 proteins (77% of the response), GRK2 (27%), and AP-2 (29%), but not GRK3. Our PKC KO cell lines expand the repertoire of KO HEK293A cell lines available to research GPCR pharmacology. Moreover, since pharmacological tools to study PKC isozymes generally lack specificity and/or potency, we present the PKC KO cell lines as more specific research tools to investigate PKC-mediated aspects of cell biology.

Significance Bioinformatic analysis revealed that approximately 40% of human messenger RNAs contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these sequences are translated, but the function of the encoded peptides remains unknown. Our study revealed a uORF encoding for a peptide exhibiting kinase inhibitory activity. This uORF, upstream of a PKC family member, possess the typical pseudosubstrate motif, which autoinhibits the catalytic activity of all PKCs. Using mouse models and human cells, we show that this peptide inhibits cancer cell survival, tumor progression, invasion, and metastasis and synergizes with chemotherapy by interfering with DNA damage response. Together, we point to a previously unrecognized function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy., Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

Simple Summary Uveal melanoma is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of several pathways which are thought to contribute to cell growth. Based on clinical and preclinical data supporting targeting of protein kinase C (PKC) and the phosphatidylinositol 3-kinase (PI3K) pathway, we conducted a phase Ib study to assess the safety of combined sotrastaurin, a PKC inhibitor, and alpelisib, a PI3K inhibitor. We found that sotrastaurin and alpelisib can be safely administered, however there was no evidence of clinical efficacy. Abstract Uveal melanoma (UM) is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of the PKC, MAPK, and PI3Kα pathways. Activity has been observed with the PKC inhibitors sotrastaurin (AEB071) and darovasertib (IDE196) in patients with UM. Inhibition of the PI3K pathway enhances PKC inhibition in in vivo models. We therefore conducted a phase Ib study of sotrastaurin in combination with the PI3Kα inhibitor alpelisib to identify a tolerable regimen that may enhance the activity of PKC inhibition alone. Patients with metastatic uveal melanoma (n = 24) or GNAQ/11 mutant cutaneous melanoma (n = 1) were enrolled on escalating dose levels of sotrastaurin (100–400 mg BID) and alpelisib (200–350 mg QD). The primary objective was to identify the maximum tolerated dose (MTD) of these agents when administered in combination. Treatment-related adverse events (AE) occurred in 86% (any grade) and 29% (Grade 3). No Grade 4–5-related AEs occurred. Dose Level 4 (sotrastaurin 200 mg BID and alpelisib 350 mg QD) was identified as the maximum tolerated dose. Pharmacokinetic analysis demonstrated increasing concentration levels with increasing doses of sotrastaurin and alpelisib, without evidence of interaction between agents. Pharmacodynamic assessment of pMARCKS and pAKT protein expression with drug exposure suggested modest target inhibition that did not correlate with clinical response. No objective responses were observed, and median progression-free survival was 8 weeks (range, 3–51 weeks). Although a tolerable dose of sotrastaurin and alpelisib was identified with pharmacodynamic evidence of target inhibition and without evidence of a corresponding immunosuppressive effect, limited clinical activity was observed.

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans. This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others.

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

Pyruvate kinase (PYK) is a speed-limited enzyme of glycolysis that catalyzes the formation of pyruvate, and plays an important role in acetyl-CoA synthesis. The acetyl-CoA is the precursor of sex pheromone biosynthesis in Helicoverpa armigera. However, the role of PYK in sex pheromone biosynthesis remains elusive. Here, PYK in H. armigera (HaPYK) was found to be highly expressed in the pheromone glands (PGs). The developmental expression profile of HaPYK was consistent with the fluctuation of sex pheromone release. Function analysis revealed that the knockdown of HaPYK led to a decrease in the levels of pyruvic acid and acetyl-CoA in PGs, which in turn caused a significant decrease in cis-11-hexadecenal (Z11-16: Ald) production, female capability to attract males, and mating frequency. Further study demonstrated that sugar feeding (5% sugar) increased the transcription and enzyme activity of HaPYK, thereby facilitating sex pheromone biosynthesis. Moreover, pheromone biosynthesis activating neuropeptide (PBAN) upregulated HaPYK activity through protein kinase C (PKC), as shown by PKC-specific inhibitor analysis. Altogether, our results revealed that PBAN activated HaPYK by Ca2+/PKC, thereby regulating the synthesis of pyruvate and subsequent acetyl-CoA, ensuring the supply of sex pheromone precursor, and finally facilitating sex pheromone biosynthesis and mating behavior.

Diabetes mellitus is a metabolic syndrome that affects millions of people worldwide. Recent studies have demonstrated that protein kinase C (PKC) activation plays an important role in hyperglycemia-induced atherosclerosis. PKC activation is involved in several cellular responses such as the expression of various growth factors, activation of signaling pathways, and enhancement of oxidative stress in hyperglycemia. However, the role of PKC activation in pro-atherogenic and anti-atherogenic mechanisms remains controversial, especially under hyperglycemic condition. In this review, we discuss the role of different PKC isoforms in lipid regulation, oxidative stress, inflammatory response, and apoptosis. These intracellular events are linked to the pathogenesis of atherosclerosis in diabetes. PKC deletion or treatment with PKC inhibitors has been studied in the regulation of atherosclerotic plaque formation and evolution. Furthermore, some preclinical and clinical studies have indicated that PKCβ and PKCδ are potential targets for the treatment of diabetic vascular complications. The current review summarizes these multiple signaling pathways and cellular responses regulated by PKC activation and the potential therapeutic targets of PKC in diabetic complications.

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., To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a 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‐kB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants reduce signaling through the nPKC and NF‐kB.

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.

Euphorbia usambarica is a traditional medicine used for gynecologic, endocrine, and urogenital illnesses in East Africa; however, its constituents and bioactivities have not been investigated. A variety of compounds isolated from Euphorbia species have been shown to have activity against latent HIV-1, the major source of HIV-1 persistence despite antiretroviral therapy. We performed bioactivity-guided isolation to identify 15 new diterpenoids (1–9, 14–17, 19, and 20) along with 16 known compounds from E. usambarica with HIV-1 latency reversal activity. Euphordraculoate C (1) exhibits a rare 6/6/3-fused ring system with a 2-methyl-2-cyclopentenone moiety. Usambariphanes A (2) and B (3) display an unusual lactone ring constructed between C-17 and C-2 in the jatrophane structure. 4β-Crotignoid K (14) revealed a 250-fold improvement in latency reversal activity compared to crotignoid K (13), identifying that configuration at the C-4 of tigliane diterpenoids is critical to HIV-1 latency reversal activity. The primary mechanism of the active diterpenoids 12–14 and 21 for the HIV-1 latency reversal activity was activation of PKC, while lignans 26 and 27 that did not increase CD69 expression, suggesting a non-PKC mechanism. Accordingly, natural constituents from E. usambarica have the potential to contribute to the development of HIV-1 eradication strategies.

Squamous cell carcinomas of the lung, head and neck, esophagus, and cervix account for more than two million cases of cancer per year worldwide with very few targetable therapies available and minimal clinical improvement in the past three decades. Although these carcinomas are differentiated anatomically, their genetic landscape shares numerous common genetic alterations. Amplification of the third chromosome’s distal portion (3q) is a distinguishing genetic alteration in most of these carcinomas and leads to copy-number gain and amplification of numerous oncogenic proteins. This area of the chromosome harbors known oncogenes involved in squamous cell fate decisions and differentiation, including TP63, SOX2, ECT2, and PIK3CA. Furthermore, novel targetable oncogenic kinases within this amplicon include PRKCI, PAK2, MAP3K13, and TNIK. TCGA analysis of these genes identified amplification in more than 20% of clinical squamous cell carcinoma samples, correlating with a significant decrease in overall patient survival. Alteration of these genes frequently co-occurs and is dependent on 3q-chromosome amplification. The dependency of cancer cells on these amplified kinases provides a route toward personalized medicine in squamous cell carcinoma patients through development of small-molecules targeting these kinases.

Due to its location, the fungal cell wall is the compartment that allows the interaction with the environment and/or the host, playing an important role during infection as well as in different biological functions such as cell morphology, cell permeability and protection against stress. All these processes involve the activation of signaling pathways within the cell. The cell wall integrity (CWI) pathway is the main route responsible for maintaining the functionality and proper structure of the cell wall. This pathway is highly conserved in the fungal kingdom and has been extensively characterized in Saccharomyces cerevisiae. However, there are still many unknown aspects of this pathway in the pathogenic fungi, such as Cryptococcus neoformans. This yeast is of particular interest because it is found in the environment, but can also behave as pathogen in multiple organisms, including vertebrates and invertebrates, so it has to adapt to multiple factors to survive in multiple niches. In this review, we summarize the components of the CWI pathway in C. neoformans as well as its involvement in different aspects such as virulence factors, morphological changes, and its role as target for antifungal therapies among others. This work was supported by Grant PID2020-114546RB-100 from the Spanish Ministry for Science and Innovation. De Oliveira H.C. was supported by Inova Fiocruz/Fundação Oswaldo Cruz. Rossi SA is funded by postdoctoral fellowship from Fundacão de Amparo á pesquisa do Estado de São Paulo (reference FAPESP-BEPE 2020/09919-0). García-Barbazán Iwas supported by a FPI fellowship (reference PRE2018-083436). Trevijano-Contador N is funded by a “Ayudas de Atracción de Talento Investigador” Contract of Community of Madrid (reference 2019-T2/BMD-14926). Sí

Cadmium (Cd) is accumulated in human astrocytes and induces the production of interleukin (IL)-6 and IL-8. Astrocytes are one of the major sources of chemokine C–C motif ligand 2 (CCL2; known as monocyte chemoattractant protein-1 [MCP-1]), in the brain. Elevated CCL2 levels are associated with cognitive impairment as well as the migration and invasion of glioblastoma cells. The present study hypothesized that non-toxic concentrations of Cd (as cadmium chloride [CdCl2]) could up-regulate CCL2 production in U-87 MG human glio-blastoma cells. The results showed that after exposure of the U-87 MG cells to CdCl2 at 1 and 10 µM, there was an up-regulation of CCL2 mRNA expression after 3 h of exposure and increased CCL2 secretion after 6 and 24 h. The study also found that inhibition of MAPK pathways, including ERK1/2, p38, and JNK by U0126, SB203580 and SP600125, respectively, reduced Cd-induced CCL2 secretion by the cells. Moreover, when cells were pretreated with Ro 32-0432 (an inhibitor of calcium-dependent PKC) and LY294002 (a PI3K inhibitor), this also resulted in a down-regulation of any Cd-induced CCL2 expression. Taken together, the results of this study allow for the conclusion to be made that CCL2 up-regulation in U-87 MG cells induced by Cd is mediated, in part, by an activation of MAPK, PI3K/Akt, and PKC pathways.

Bisindolylmaleimide I (BIM-I) is a competitive pan protein kinase C inhibitor with anti-inflammatory and anti-metastatic properties, suggested to treat inflammatory diseases and various cancer entities. However, despite its therapeutic potential, BIM-I has two major drawbacks, i.e., it has a poor water solubility, and it binds the human ether-à-go-go-related gene (hERG) ion channels, potentially causing deadly arrhythmias. In this case, a targeted delivery of BIM-I is imperative to minimize peripheral side effects. To circumvent these drawbacks BIM-I was encapsulated into nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) functionalized by the near-infrared dye DY-635. DY-635 served as an active targeting moiety since it selectively binds the OATP1B1 and OATP1B3 transporters that are highly expressed in liver and cancer cells. PLGA-DY-635 (BIM-I) nanoparticles were produced by nanoprecipitation and characterized using dynamic light scattering, analytical ultracentrifugation, and cryogenic transmission electron microscopy. Particle sizes were found to be in the range of 20 to 70 nm, while a difference in sizes between the drug-loaded and unloaded particles was observed by all analytical techniques. In vitro studies demonstrated that PLGA-DY-635 (BIM-I) NPs prevent the PKC activation efficiently, proving the efficacy of the inhibitor after its encapsulation, and suggesting that BIM-I is released from the PLGA-NPs. Ultimately, our results present a feasible formulation strategy that improved the cytotoxicity profile of BIM-I and showed a high cellular uptake in the liver as demonstrated in vivo by intravital microscopy investigations.

Cordyceps militaris has been widely studied for its various pharmacological activities such as antitumor, anti-inflammation, and immune regulation. The binding of an allergen to IgE-sensitized mast cells in nasal mucosa triggers allergic rhinitis. We found that oral administration of 300 mg/kg of the ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruiting bodies significantly alleviated the symptoms of ovalbumin-induced allergic rhinitis in mice, including sneeze/scratch, mast cell activation, eosinophil infiltration, and Syk activation. The treatment of ethanol extract significantly suppressed the release of β-hexosaminidase (a degranulation marker) and mRNA expression levels of various cytokines, including IL-3, IL-10, and IL-13 in activated RBL2H3 cells. The ethanol extract and β-sitostenone, which was purified from the extract, could respectively reduce the Ca2+ ion mobilization in activated RBL-2H3 cells. Furthermore, results collected from western immunoblotting demonstrated that ethanol extract significantly retarded Ca2+ ion mobilization-initiated signaling cascade, which provoked the expression of various allergic cytokines. Also, the extract incubation interfered with P38 as well as NF-kB activation and Nrf-2 translocation. Our study suggested that ethanol extract possessed some natural constituents which could inhibit immediate degranulation and de novo synthesis of allergic cytokines via inhibition of Ca2+ ion mobilization in mast cells in the nasal mucosa of allergic rhinitis mice.

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 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.

BackgroundSeveral lines of evidence suggest the abnormalities of protein kinase C (PKC) signaling system in mood disorders and suicide based primarily on the studies of PKC and its isozymes in the platelets and postmortem brain of depressed and suicidal subjects. In this study, we examined the role of PKC isozymes in depression and suicide.MethodsWe determined the protein and mRNA expression of various PKC isozymes in the prefrontal cortical region (Brodmann area 9) in 24 normal control subjects, 24 depressed suicide (DS) subjects, and 12 depressed nonsuicide (DNS) subjects. The levels of mRNA in the prefrontal cortex were determined by quantitative real-time reverse transcription PCR, and the protein expression was determined by western blotting.ResultsWe observed a significant decrease in mRNA expression of PKCα, PKCβI, PKCδ, and PKCε and decreased protein expression in either the membrane or the cytosol fraction of PKC isozymes PKCα, PKCβI, PKCβII, and PKCδ in DS and DNS subjects compared with normal control subjects.ConclusionsThe current study provides detailed evidence of specific dysregulation of certain PKC isozymes in the postmortem brain of DS and DNS subjects and further supports earlier evidence for the role of PKC in the platelets and brain of the adult and teenage depressed and suicidal population. This comprehensive study may lead to further knowledge of the involvement of PKC in the pathophysiology of depression and suicide.

In addition to antigen presentation to CD4+ T cells, aggregation of cell surface major histocompatibility complex class II (MHC-II) molecules induces signal transduction in antigen presenting cells that regulate cellular functions. We previously reported that crosslinking of MHC-II induced the endocytosis of MHC-II, which was associated with decreased surface expression levels in murine dendritic cells (DCs) and resulted in impaired activation of CD4+ T cells. However, the downstream signal that induces MHC-II endocytosis remains to be elucidated. In this study, we found that the crosslinking of MHC-II induced intracellular Ca2+ mobilization, which was necessary for crosslinking-induced MHC-II endocytosis. We also found that these events were suppressed by inhibitors of Syk and phospholipase C (PLC). Treatments with a phorbol ester promoted MHC-II endocytosis, whereas inhibitors of protein kinase C (PKC) suppressed crosslinking-induced endocytosis of MHC-II. These results suggest that PKC could be involved in this process. Furthermore, crosslinking-induced MHC-II endocytosis was suppressed by inhibitors of clathrin-dependent endocytosis. Our results indicate that the crosslinking of MHC-II could stimulate Ca2+ mobilization and induce the clathrin-dependent endocytosis of MHC-II in murine DCs.

Trimeric G proteins play a central role in the G protein signaling in filamentous fungi and Gα subunits are the major component of trimeric G proteins. In this study, we characterize three Gα subunits in the human pathogen Aspergillus fumigatus. While the deletion of gpaB and ganA led to reduced colony growth, the growth of the ΔgpaA strain was increased in minimal media. The germination rate, conidiation, and mRNA expression of key asexual development regulators were significantly decreased by the loss of gpaB. In contrast, the deletion of gpaA resulted in increased conidiation and mRNA expression levels of key asexual regulators. The deletion of gpaB caused a reduction in conidial tolerance against H2O2, but not in paraquat (PQ). Moreover, the ΔgpaB mutant showed enhanced susceptibility against membrane targeting azole antifungal drugs and reduced production of gliotoxin (GT). The protein kinase A (PKA) activity of the ΔganA strain was severely decreased and protein kinase C (PKC) activity was detected all strains at similar levels, indicating that all G protein α subunits of A. fumigatus may be a component of the cAMP/PKA signaling pathway and appear to possess the PKC signaling pathway as an alternative backup pathway to compensate for PKA depletion. Collectively, the three Gα subunits regulate growth, germination, asexual development, resistance to oxidative stress, and GT production differently via the PKA or PKC signaling pathway. The function of GanA of A. fumigatus was elucidated for the first time.

Protein kinase C (PKC) and Protein kinase D (PKD) isoforms can sense diacylglycerol (DAG) generated in the different cellular compartments in various physiological processes. DAG accumulates in multiple organs of the obese subjects, which leads to the disruption of metabolic homeostasis and the development of diabetes as well as associated diseases. Multiple studies proved that aberrant activation of PKCs and PKDs contributes to the development of metabolic diseases. DAG-sensing PKC and PKD isoforms play a crucial role in the regulation of metabolic homeostasis and therefore might serve as targets for the treatment of metabolic disorders such as obesity and diabetes.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by the proliferation of immature myeloid cells, with impaired differentiation and maturation. Pyruvate dehydrogenase kinase (PDK) is a pyruvate dehydrogenase complex (PDC) phosphatase inhibitor that enhances cell glycolysis and facilitates tumor cell proliferation. Inhibition of its activity can induce apoptosis of tumor cells. Currently, little is known about the role of PDKs in AML. Therefore, we screened The Cancer Genome Atlas (TCGA) database for de novo AML patients with complete clinical information and PDK family expression data, and 84 patients were included for the study. These patients did not undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Univariate analysis showed that high expression of PDK2 was associated with shorter EFS (P = 0.047), and high expression of PDK3 was associated with shorter OS (P = 0.026). In multivariate analysis, high expression of PDK3 was an independent risk factor for EFS and OS (P 0.05). Our results indicated that high expressions of PDK2 and PDK3, especially the latter, were poor prognostic factors of AML, and the effect could be overcome by allo-HSCT.

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

Diabetic nephropathy (DN) is the leading cause of the end-stage renal disease worldwide and an independent risk factor for cardiovascular mortalities in diabetic patients. We evaluated the anti-hyperglycemic and anti-chronic renal damage effect of mulberry leaf tea, a potential functional food material. Mulberry leaf tea significantly alleviated symptoms of DN rats including reducing blood glucose level, improving insulin sensibility and ameliorating blood lipid profile and chronic renal damages. The abundant pharmacologically compounds in mulberry leaf tea such as 1-DNJ, flavonoids, polyphenols and polysaccharides were likely responsible for its beneficial effects. Furthermore, we found that mulberry leaf tea alleviated DN by reducing DAG-PKC cascades including regulating energy metabolism, improving insulin sensibility and reducing inflammatory response. Mulberry leaf tea also modulated the community structure of the intestinal flora which were closely associated with diabetes and its complications. Mulberry leaf tea provides a novel dietotherapy for DN.

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.

A better understanding of the link between cellular metabolism and tumorigenesis is needed. Here, we report characterization of a novel protein named coiled-coil helix tumor and metabolism 1 (CHTM1). We have found that CHTM1 is associated with cancer and cellular metabolism. CHTM1 localizes to mitochondria and cytosol, and its deficiency in cancer cells results in decreased mitochondrial oxygen consumption and ATP levels as well as oxidative stress indicating mitochondrial dysfunction. CHTM1-deficient cancer cells display poor growth under glucose/glutamine-deprived conditions, whereas cells expressing increased levels of exogenous CHTM1 exhibit enhanced proliferation and survival under similar conditions. CHTM1 deficiency also leads to defects in lipid metabolism resulting in fatty acid accumulation, which explains poor growth of CHTM1-deficient cells under glucose/glutamine deprivation since nutrient deprivation increases dependency on lipids for energy generation. We also demonstrate that CHTM1 mediates its effect via the PKC, CREB, and PGC-1alpha signaling axis, and cytosolic accumulation of CHTM1 during nutrient deprivation appears to be important for its effect on cellular signaling events. Furthermore, analyses of tissue specimens from 71 breast and 97 colon cancer patients show CHTM1 expression to be upregulated in the majority of tumor specimens representing these malignancies. Collectively, our findings are highly significant because CHTM1 is a novel metabolic marker that is important for the growth of tumorigenic cells under limiting nutrient supplies and thus, links cellular metabolism and tumorigenesis.

Long-term depression (LTD) and long-term potentiation (LTP) in the cerebellum are important for motor learning. However, the signaling mechanisms controlling whether LTD or LTP is induced in response to synaptic stimulation remain obscure. Using a unified model of LTD and LTP at the cerebellar parallel fiber-Purkinje cell (PF-PC) synapse, we delineate the coordinated pre- and postsynaptic signaling that determines the direction of plasticity. We show that LTP is the default response to PF stimulation above a well-defined frequency threshold. However, if the calcium signal surpasses the threshold for CaMKII activation, then an ultrasensitive “on switch” activates an extracellular signal-regulated kinase (ERK)-based positive feedback loop that triggers LTD instead. This postsynaptic feedback loop is sustained by another, trans-synaptic, feedback loop that maintains nitric oxide production throughout LTD induction. When full depression is achieved, an automatic “off switch” inactivates the feedback loops, returning the network to its basal state and demarcating the end of the early phase of LTD.

International audience; Diclofenac (DCF) adverse reactions involve diverse mechanisms in different models. We recently demonstrated that DCF-induced toxicity in HepaRG decreases as they express DCF-metabolizing enzymes. DCF metabolism promotes toxicity in Saccharomyces cerevisiae expressing heterologous cytochromes-P450. N-Acetylcysteine (NAC) is used to treat diverse medical conditions due to its multiple properties (antioxidant, metal chelator, thiol-disulfide disruption). The latter property accounts for its mucolytic effects and broadens its potential molecular targets to signal transduction proteins, ABC transporters and others. Interaction of NAC with DCF effects depends on the experimental model. This study aims to investigate NAC/DCF interaction and the involvement of ABC transporters in wild type and mutant Saccharomyces cerevisiae. DCF inhibited yeast growth in a dose- and time-dependent manner and the cells started adapting to DCF 24-h post-treatment. NAC potentiated DCF-induced toxicity if added prior or parallel to DCF. Pretreatment with NAC increased its potentiation effect and compromised cells adaption to DCF. Post-treatment with NAC potentiated DCF toxicity without compromising adaptation. Moreover, mutant strains in ABC transporters Pdr5, Yor1, Bpt1 or Pdr15, were more sensitive to DCF; while mutant strains in Pdr5, Vmr1 or Pdr12 were more sensitive to NAC/DCF interaction. DCF ± NAC elicited on the mutant strain in Yap1, an oxidative stress-related protein, the same effects as on the wild type. Therefore, oxidative stress does not seem to be key actor in DCF toxicity in our model. Our hypothesis is that NAC potentiation effect is at least due to its ability to disrupt disulfide bridge in proteins required to overcome DCF toxicity in yeast.

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 shRNA 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 shRNA 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 shRNA. 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.

Recent evidence has shown the cardioprotective effect of PDE5 inhibition in myocardial ischemia/reperfusion injury, heart failure and cardiac hypertrophy. To investigate the biochemical changes that occur during PDE5 inhibition in cardiac cells, this study assessed the metabolic profile of the HL1 cell line, a murine atrial cell line with adult cardiomyocyte properties. After one hour of treatment with sildenafil, glycolysis was moderately but selectively stimulated, unlike the pentose phosphate pathway and the Krebs cycle. Moreover, malate and a-Ketoglutarate accumulated, paralleled by a decrease in aspartate and glutamate. Interestingly, increased activity of malate dehydrogenase (MDH) was also detected in these cells after sildenafil treatment. Thus, we hypothesized that sildenafil stimulates the malate-aspartate shuttle (MAS) with the final effect of transferring electrons and protons from glycolysis-derived cytosolic NADH into the matrix for use by the electron transport chain, using malate as an electron carrier. Through this metabolic modification, sildenafil may counteract what is often observed in ischemia, i.e. reduced MAS flux as well as a dramatic acceleration of glycolysis, which switches to lactate production. Additionally, the results observed in HL1 cells were also found in isolated mouse hearts. The documented metabolic alteration in cardiomyocytes upon treatment with sildenafil occurred by stimulating cGMP production, which did not activate PKG (cGMP-PKG signaling), since the addition of DT-2, a PKG inhibitor, did not block malate accumulation and increased MDH activity. Conversely, the addition of chelerythrine, a PKC inhibitor, counteracted both malate accumulation and MAS activation, supporting previous evidence that, upon the addition of sildenafil, some PKC isoforms may be implicated in cardioprotection (cGMP-PKC signaling). Interestingly, an increase in cGMP, driven by sildenafil, another cGMP stimulator such as nitroprusside (SNP), or a C-type natriuretic peptide (CNP) which does not inhibit PDE5, led to MAS stimulation and increased MDH activity.

A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions. Also, the knowledge is expected to facilitate devising innovative strategies that more precisely monitor and normalize organ functions by targeting individual smooth muscles. Evidence has established a current paradigm that the myosin light chain phosphatase (MLCP) is a master regulator of smooth muscle responsiveness to stimuli. Cellular MLCP activity is negatively and positively regulated in response to G-protein activation and cAMP/cGMP production, respectively, through the MYPT1 regulatory subunit and an endogenous inhibitor protein named CPI-17. In this article we review the outcomes from two decade of research on the CPI-17 signaling and discuss emerging paradoxes in the view of signaling pathways regulating smooth muscle functions through MLCP.

Ischemic postconditioning (IPoC) reduces reperfusion arrhythmias but the antiarrhythmic mechanisms remain unknown. The aim of this study was to analyze IPoC electrophysiological effects and the role played by adenosine A1, A2A and A3 receptors, protein kinase C, ATP-dependent potassium (KATP) channels, and connexin 43. IPoC reduced reperfusion arrhythmias (mainly sustained ventricular fibrillation) in isolated rat hearts, an effect associated with a transient delay in epicardial electrical activation, and with action potential shortening. Electrical impedance measurements and Lucifer-Yellow diffusion assays agreed with such activation delay. However, this delay persisted during IPoC in isolated mouse hearts in which connexin 43 was replaced by connexin 32 and in mice with conditional deletion of connexin 43. Adenosine A1, A2A and A3 receptor blockade antagonized the antiarrhythmic effect of IPoC and the associated action potential shortening, whereas exogenous adenosine reduced reperfusion arrhythmias and shortened action potential duration. Protein kinase C inhibition by chelerythrine abolished the protective effect of IPoC but did not modify the effects on action potential duration. On the other hand, glibenclamide, a KATP inhibitor, antagonized the action potential shortening but did not interfere with the antiarrhythmic effect. The antiarrhythmic mechanisms of IPoC involve adenosine receptor activation and are associated with action potential shortening. However, this action potential shortening is not essential for protection, as it persisted during protein kinase C inhibition, a maneuver that abolished IPoC protection. Furthermore, glibenclamide induced the opposite effects. In addition, IPoC delays electrical activation and electrical impedance recovery during reperfusion, but these effects are independent of connexin 43.

Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α–converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA–mediated knockdown, and overexpression of dominant-negative PKCβII variants indicated that PKCβII is a major player in JNK inhibition– and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCβII and tumor necrosis factor-α–converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCβII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.