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Frontiers in Cell and Developmental... 2022Protein kinase C (PKC) isozymes transduce myriad signals within the cell in response to the generation of second messengers from membrane phospholipids. The conventional... (Review)
Review
Protein kinase C (PKC) isozymes transduce myriad signals within the cell in response to the generation of second messengers from membrane phospholipids. The conventional isozyme PKCγ reversibly binds Ca and diacylglycerol, which leads to an open, active conformation. PKCγ expression is typically restricted to neurons, but evidence for its expression in certain cancers has emerged. PKC isozymes have been labeled as oncogenes since the discovery that they bind tumor-promoting phorbol esters, however, studies of cancer-associated PKC mutations and clinical trial data showing that PKC inhibitors have worsened patient survival have reframed PKC as a tumor suppressor. Aberrant expression of PKCγ in certain cancers suggests a role outside the brain, although whether PKCγ also acts as a tumor suppressor remains to be established. On the other hand, PKCγ variants associated with spinocerebellar ataxia type 14 (SCA14), a neurodegenerative disorder characterized by Purkinje cell degeneration, enhance basal activity while preventing phorbol ester-mediated degradation. Although the basis for SCA14 Purkinje cell degeneration remains unknown, studies have revealed how altered PKCγ activity rewires cerebellar signaling to drive SCA14. Importantly, enhanced basal activity of SCA14-associated mutants inversely correlates with age of onset, supporting that enhanced PKCγ activity drives SCA14. Thus, PKCγ activity should likely be inhibited in SCA14, whereas restoring PKC activity should be the goal in cancer therapies. This review describes how PKCγ activity can be lost or gained in disease and the overarching need for a PKC structure as a powerful tool to predict the effect of PKCγ mutations in disease.
PubMed: 35800893
DOI: 10.3389/fcell.2022.929510 -
Frontiers in Immunology 2022Cell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of... (Observational Study)
Observational Study
BACKGROUND
Cell-based functional immune-assays may allow for risk stratification of patients with complex, heterogeneous immune disorders such as sepsis. Given the heterogeneity of patient responses and the uncertain immune pathogenesis of sepsis, these assays must first be defined and calibrated in the healthy population.
OBJECTIVE
Our objective was to compare the internal consistency and practicality of two immune assays that may provide data on surrogate markers of the innate and adaptive immune response. We hypothesized that a rapid turnaround, microfluidic-based immune assay (ELLA) would be comparable to a dual-color, enzyme-linked immunospot (ELISpot) assay in identifying tumor necrosis factor (TNF) and interferon (IFN)γ production following whole blood stimulation.
DESIGN
This was a prospective, observational cohort analysis. Whole blood samples from ten healthy, immune-competent volunteers were stimulated for either 4 hours or 18 hours with lipopolysaccharide, anti-CD3/anti-CD28 antibodies, or phorbol 12-myristate 13-acetate with ionomycin to interrogate innate and adaptive immune responses, respectively.
MEASUREMENTS AND MAIN RESULTS
ELLA analysis produced more precise measurement of TNF and IFNγ concentrations as compared with ELISpot, as well as a four- to five-log dynamic range for TNF and IFNγ concentrations, as compared with a two-log dynamic range with ELISpot. Unsupervised clustering accurately predicted the immune stimulant used for 90% of samples analyzed ELLA, as compared with 72% of samples analyzed ELISpot.
CONCLUSIONS
We describe, for the first time, a rapid and precise assay for functional interrogation of the innate and adaptive arms of the immune system in healthy volunteers. The advantages of the ELLA microfluidic platform may represent a step forward in generating a point-of-care test with clinical utility, for identifying deranged immune phenotypes in septic patients.
Topics: Cytokines; Enzyme-Linked Immunospot Assay; Humans; Interferon-gamma; Prospective Studies; Sepsis; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha
PubMed: 35860253
DOI: 10.3389/fimmu.2022.940030 -
Biomolecules & Therapeutics Jul 2018In this study, we investigated the effects of pelargonidin, an anthocyanidin found in many fruits and vegetables, on endothelium-independent vascular contractility to...
In this study, we investigated the effects of pelargonidin, an anthocyanidin found in many fruits and vegetables, on endothelium-independent vascular contractility to determine the underlying mechanism of relaxation. Isometric contractions of denuded aortic muscles from male rats were recorded, and the data were combined with those obtained in western blot analysis. Pelargonidin significantly inhibited fluoride-, thromboxane A2-, and phorbol ester-induced vascular contractions, regardless of the presence or absence of endothelium, suggesting a direct effect of the compound on vascular smooth muscles via a different pathway. Pelargonidin significantly inhibited the fluoride-dependent increase in the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation at Thr-855 and the phorbol 12,13-dibutyrate-dependent increase in the level of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation at Thr202/Tyr204, suggesting the inhibition of Rho-kinase and mitogen-activated protein kinase kinase (MEK) activities and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxation effect of pelargonidin on agonist-dependent vascular contractions includes inhibition of Rho-kinase and MEK activities, independent of the endothelial function.
PubMed: 29390250
DOI: 10.4062/biomolther.2017.197 -
Cellular Signalling Dec 2017Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays...
Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays a critical role in hematologic cell signaling, whereas Vav2/3 have a wider tissue distribution, but all 3 Vav proteins are implicated in cancer development. A structural feature of Vav1/2/3 is the presence of an atypical C1 domain, which possesses close structural homology to the typical C1 domains of protein kinase C but which fails to bind the second messenger diacylglycerol or the potent analogs, the phorbol esters. Previously, we have shown that five residues in the Vav1 C1 domain are responsible for its lack of phorbol ester binding. Here, we show that the lack of phorbol ester binding of Vav3 has a similar basis. We then explore the consequences of phorbol ester binding to a modified Vav3 in which the C1 domain has been altered to allow phorbol ester binding. We find both disruption of the guanyl nucleotide exchange activity of the modified Vav 3 as well as a shift in localization to the membrane upon phorbol ester treatment. This change in localization is associated with altered interactions with other signaling proteins. The studies provide a first step in assessing the potential for the design of custom C1 domain targeted molecules selective for the atypical C1 domains of Vav family proteins.
Topics: Amino Acid Sequence; Humans; Molecular Targeted Therapy; Neoplasms; Phorbol Esters; Protein Domains; Protein Kinase C; Protein Structure, Tertiary; Proto-Oncogene Proteins c-vav; Signal Transduction
PubMed: 28927664
DOI: 10.1016/j.cellsig.2017.09.008 -
AIDS Research and Human Retroviruses Jan 2015Highly active antiretroviral therapy (HAART) is very effective in suppressing HIV-1 replication and restoring immune functions in HIV-infected individuals. However, it... (Review)
Review
Highly active antiretroviral therapy (HAART) is very effective in suppressing HIV-1 replication and restoring immune functions in HIV-infected individuals. However, it fails to eradicate the latent viral reservoirs and fully resolve chronic inflammation in HIV infection. The "shock-and-kill" strategy was recently proposed to induce latent HIV expression in the presence of HAART. Recent studies have shown that the protein kinase C (PKC) agonists are highly potent in inducing latent HIV expression from the viral reservoirs in vitro and ex vivo and in protecting primary CD4(+) T cells from HIV infection through down-modulation of their HIV coreceptor expression. The PKC agonists are excellent candidates for advancing to clinical HIV eradication strategies. This article will present a critical review of the structure and function of known PKC agonists, their mechanisms for the reactivation of latent HIV expression, and the potential of these compounds for advancing clinical HIV eradication strategies.
Topics: Anti-HIV Agents; Antiretroviral Therapy, Highly Active; CD4-Positive T-Lymphocytes; Enzyme Activation; HIV Infections; HIV-1; Humans; NF-kappa B p50 Subunit; NF-kappa B p52 Subunit; Phorbol Esters; Protein Binding; Protein Kinase C; Proto-Oncogene Proteins c-rel; Receptors, HIV; Transcription Factor RelA; Transcription Factor RelB; Virus Latency
PubMed: 25287643
DOI: 10.1089/AID.2014.0199 -
Phytomedicine : International Journal... May 2015The root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk...
Dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppressed the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.
BACKGROUND
The root of Asparagus cochinchinensis (Lour.) Merr. has been utilized as mucoregulators and expectorants for controlling the airway inflammatory diseases in folk medicine.
HYPOTHESIS/PURPOSE
We investigated whether dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis (Lour.) Merr. suppress the gene expression and production of airway MUC5AC mucin induced by phorbol ester and growth factor.
STUDY DESIGN
Confluent NCI-H292 cells were pretreated with dioscin or methylprotodioscin for 30 min and then stimulated with EGF or PMA for 24 h. The MUC5AC mucin gene expression was measured by RT-PCR. Production of MUC5AC mucin protein was measured by ELISA.
RESULTS
(1) Dioscin and methylprotodioscin suppressed the expression of MUC5AC mucin gene induced by EGF or PMA; (2) dioscin suppressed the production of MUC5AC mucin induced by either EGF at 10(-5) M (p < 0.05) and 10(-6) M (p < 0.05) or PMA at 10(-4) M (p < 0.05), 10(-5) M (p < 0.05) and 10(-6) M (p < 0.05); (3) methylprotodioscin also suppressed the production of MUC5AC mucin induced by either EGF at 10(-4) M (p < 0.05) or PMA at 10(-4) M (p < 0.05).
CONCLUSION
These results suggest that dioscin and methylprotodioscin isolated from the root of Asparagus cochinchinensis suppress the gene expression and production of MUC5AC mucin, by directly acting on airway epithelial cells, and the results are consistent with the traditional use of Asparagus cochinchinensis as remedy for diverse inflammatory pulmonary diseases.
Topics: Asparagus Plant; Cell Line, Tumor; Diosgenin; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Mucin 5AC; Plant Roots; Saponins; Tetradecanoylphorbol Acetate
PubMed: 25981923
DOI: 10.1016/j.phymed.2015.03.009 -
Journal of Agricultural and Food... May 2015Although flavonoids have been reported for their benefits and nutraceutical potential use, the importance of their structure on their beneficial effects, especially on...
Number of Hydroxyl Groups on the B-Ring of Flavonoids Affects Their Antioxidant Activity and Interaction with Phorbol Ester Binding Site of PKCδ C1B Domain: In Vitro and in Silico Studies.
Although flavonoids have been reported for their benefits and nutraceutical potential use, the importance of their structure on their beneficial effects, especially on signal transduction mechanisms, has not been well clarified. In this study, three flavonoids, pinocembrin, naringenin, and eriodictyol, were chosen to determine the effect of hydroxyl groups on the B-ring of flavonoid structure on their antioxidant activity. In vitro assays, including DPPH scavenging activity, ROS quantification by flow cytometer, and proteins immunoblotting, and in silico analysis by molecular docking between the flavonoids and C1B domain of PKCδ phorbol ester binding site were both used to complete this study. Eriodictyol (10 μM), containing two hydroxyl groups on the B-ring, exhibited significantly higher (p < 0.05) antioxidant activity than pinocembrin and naringenin. The IC50 values of eriodictyol, naringenin, and pinocembrin were 17.4 ± 0.40, 30.2 ± 0.61, and 44.9 ± 0.57 μM, respectively. In addition, eriodictyol at 10 μM remarkably inhibited the phosphorylation of PKCδ at 63.4% compared with PMA-activated RAW264.7, whereas pinocembrin and naringenin performed inhibition activity at 76.8 and 72.6%, respectively. According to the molecular docking analysis, pinocembrin, naringenin, and eriodictyol showed -CDOCKER_energy values of 15.22, 16.95, and 21.49, respectively, reflecting that eriodictyol could bind with the binding site better than the other two flavonoids. Interestingly, eriodictyol had a remarkably different pose to bind with the kinase as a result of the two hydroxyl groups on its B-ring, which consequently contributed to greater antioxidant activity over pinocembrin and naringenin.
Topics: Animals; Antioxidants; Binding Sites; Cell Line; Cell Survival; Computer Simulation; Flavonoids; Mice; Molecular Docking Simulation; Molecular Structure; Oxidative Stress; Phorbol Esters; Phosphorylation; Protein Binding; Protein Kinase C-delta; Protein Structure, Tertiary
PubMed: 25907027
DOI: 10.1021/acs.jafc.5b00312 -
Trends in Pharmacological Sciences May 2017The discovery in the 1980s that protein kinase C (PKC) is a receptor for the tumor-promoting phorbol esters fueled the dogma that PKC is an oncoprotein. Yet 30+ years of... (Review)
Review
The discovery in the 1980s that protein kinase C (PKC) is a receptor for the tumor-promoting phorbol esters fueled the dogma that PKC is an oncoprotein. Yet 30+ years of clinical trials for cancer using PKC inhibitors not only failed, but in some instances worsened patient outcome. The recent analysis of cancer-associated mutations, from diverse cancers and throughout the PKC family, revealed that PKC isozymes are generally inactivated in cancer, supporting a tumor suppressive function. In keeping with a bona fide tumor suppressive role, germline causal loss-of-function (LOF) mutations in one isozyme have recently been identified in lymphoproliferative disorders. Thus, strategies in cancer treatment should focus on restoring rather than inhibiting PKC.
Topics: Animals; Genes, Tumor Suppressor; Germ-Line Mutation; Humans; Neoplasms; Protein Kinase C
PubMed: 28283201
DOI: 10.1016/j.tips.2017.02.002 -
International Journal of Molecular... Oct 2022We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide...
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.
Topics: Mice; Animals; Vasoactive Intestinal Peptide; Cyclic AMP Response Element-Binding Protein; Suprachiasmatic Nucleus Neurons; Suprachiasmatic Nucleus; Protein Kinase C; Luciferases; Phorbol Esters
PubMed: 36293078
DOI: 10.3390/ijms232012226 -
Archives of Biochemistry and Biophysics Jul 2020Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in...
Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.
Topics: Amino Acid Sequence; Butadienes; Cell Line, Tumor; Egtazic Acid; Humans; Indoles; Isoquinolines; Maleimides; Nitriles; Protein Conformation, alpha-Helical; Protein Kinase C; Protein Structure, Tertiary; RNA, Messenger; Receptors, G-Protein-Coupled; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Up-Regulation
PubMed: 32339486
DOI: 10.1016/j.abb.2020.108375