-
Cell Cycle (Georgetown, Tex.) Mar 2022Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell...
Exocytosis of large dense core vesicles is responsible for hormone secretion in neuroendocrine cells. The population of primed vesicles ready to release upon cell excitation demonstrates large heterogeneity. However, there are currently no models that clearly reflect such heterogeneity. Here, we develop a novel model based on single vesicle release events from amperometry recordings of PC12 cells using carbon fiber microelectrode. In this model, releasable vesicles can be grouped into two subpopulations, namely, SP1 and SP2. SP1 vesicles replenish quickly, with kinetics of ~0.0368 s, but likely undergo slow fusion pore expansion (amperometric signals rise at ~2.5 pA/ms), while SP2 vesicles demonstrate slow replenishment (kinetics of ~0.0048 s) but prefer fast dilation of fusion pore, with an amperometric signal rising rate of ~9.1 pA/ms. Phorbol ester enlarges the size of SP2 partially via activation of protein kinase C and conveys SP1 vesicles into SP2. Inhibition of Rho GTPase-dependent actin rearrangement almost completely depletes SP2. We also propose that the phorbol ester-sensitive vesicle subpopulation (SP2) is analogous to the subset of superprimed synaptic vesicles in neurons. This model provides a meticulous description of the architecture of the readily releasable vesicle pool and elucidates the heterogeneity of the vesicle priming mechanism.
Topics: Animals; Dense Core Vesicles; Exocytosis; PC12 Cells; Phorbol Esters; Rats; Synaptic Vesicles
PubMed: 35067177
DOI: 10.1080/15384101.2022.2026576 -
International Journal of Molecular... Sep 2021Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve...
Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.
Topics: Acetophenones; Cell Differentiation; Cell Proliferation; Cell Shape; Cell Survival; Electron Spin Resonance Spectroscopy; Free Radicals; HL-60 Cells; Humans; Hydroxyl Radical; Monocytes; NADP; Proteins; Staining and Labeling; Tetradecanoylphorbol Acetate; U937 Cells
PubMed: 34576127
DOI: 10.3390/ijms22189963 -
Journal of Cell Science Feb 2021KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However,...
KRIT1 is a scaffolding protein that regulates multiple molecular mechanisms, including cell-cell and cell-matrix adhesion, and redox homeostasis and signaling. However, rather little is known about how KRIT1 is itself regulated. KRIT1 is found in both the cytoplasm and the nucleus, yet the upstream signaling proteins and mechanisms that regulate KRIT1 nucleocytoplasmic shuttling are not well understood. Here, we identify a key role for protein kinase C (PKC) in this process. In particular, we found that PKC activation promotes the redox-dependent cytoplasmic localization of KRIT1, whereas inhibition of PKC or treatment with the antioxidant N-acetylcysteine leads to KRIT1 nuclear accumulation. Moreover, we demonstrated that the N-terminal region of KRIT1 is crucial for the ability of PKC to regulate KRIT1 nucleocytoplasmic shuttling, and may be a target for PKC-dependent regulatory phosphorylation events. Finally, we found that silencing of PKCα, but not PKCδ, inhibits phorbol 12-myristate 13-acetate (PMA)-induced cytoplasmic enrichment of KRIT1, suggesting a major role for PKCα in regulating KRIT1 nucleocytoplasmic shuttling. Overall, our findings identify PKCα as a novel regulator of KRIT1 subcellular compartmentalization, thus shedding new light on the physiopathological functions of this protein.
Topics: Active Transport, Cell Nucleus; HeLa Cells; Humans; KRIT1 Protein; Phosphorylation; Protein Kinase C-alpha; Tetradecanoylphorbol Acetate
PubMed: 33443102
DOI: 10.1242/jcs.250217 -
The Journal of Biological Chemistry Oct 2021DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ)....
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify phosphorylation targets of DNA-PKcs. Our results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at serine 301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules such as IL (interleukin) 2, IL6, IFNγ, and NFκB. Inhibition of DNA-PKcs by treatment with a DNA-PKcs specific inhibitor NU7441 or shRNA knockdown increased proteasomal degradation of Egr1. Mutation of serine 301 to alanine via CRISPR-Cas9 reduced EGR1 protein expression and decreased Egr1-dependent transcription of IL2 in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.
Topics: Animals; Cytokines; DNA-Activated Protein Kinase; DNA-Binding Proteins; Early Growth Response Protein 1; Humans; Jurkat Cells; Lymphocyte Activation; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, SCID; Mutation, Missense; Protein Stability; T-Lymphocytes; Transcription, Genetic; Mice
PubMed: 34562454
DOI: 10.1016/j.jbc.2021.101209 -
Immunologic Research Oct 2022Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators...
Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)-mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)-differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC-induced mRNA expression of interferon (IFN)-stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-β mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.
Topics: Anti-Inflammatory Agents; Humans; Interferon Regulatory Factor-3; Interferon-beta; Janus Kinases; Ligands; Lipopolysaccharides; Poly I-C; Quinazolines; RNA, Messenger; STAT1 Transcription Factor; THP-1 Cells; Tetradecanoylphorbol Acetate; Toll-Like Receptor 3
PubMed: 35666435
DOI: 10.1007/s12026-022-09301-z -
Folia Neuropathologica 2020Nuclear factor-kB (NF-kB) is a critical regulator of inflammatory responses following intracerebral haemorrhage (ICH). According to our previous study, inhibiting the...
Separate administration of ammonium pyrrolidinedithiocarbamate and phorbol myristate acetate at early and late stages decreases secondary brain injury following intracerebral haemorrhage in rats via the NF-κB pathway.
INTRODUCTION
Nuclear factor-kB (NF-kB) is a critical regulator of inflammatory responses following intracerebral haemorrhage (ICH). According to our previous study, inhibiting the p65 subunit at an early stage after ICH can reduce cell death, while inhibiting c-Rel at a late stage can lead to the opposite result. The aim of this study is to clarify whether patient prognosis can be improved by inhibiting p65 at the early stage and promoting c-Rel at the late stage.
MATERIAL AND METHODS
Rats were divided into a sham group, ICH group, early NF-kB-inhibiting group using ammonium pyrrolidinedithiocarbamate (PDTC; group A, p65 subunit was dominant and inhibited at the early stage), late NF-kB-activating group using phorbol myristate acetate (PMA; group B, c-Rel was dominant and promoted at the late stage), and early NF-kB-inhibiting and late-activating group (group C, p65 subunit was inhibited at the early stage and c-Rel was promoted at the late stage). At preset time points after ICH, perihematomal tissue was obtained for detection of NF-kB activation, cell death, and expression of caspase-3, Bcl-2, and NF-kB subunits, to evaluate of the effect of PDTC and PMA.
RESULTS
At four days after ICH, p65 expression (p < 0.01) and the number of TUNEL-positive cells (p < 0.01) in group A were significantly lower than in the ICH group. At 10 days after ICH, c-Rel expression in groups B and C was significantly higher than in other groups (p < 0.01 for all). TUNEL-positive cell numbers in groups A and B were significantly lower than in the ICH group, though more numerous than in group C (p < 0.01 for all).
CONCLUSIONS
Administration of both PDTC at the early stage and PMA at the late stage reduced perihematomal cell death after ICH, and using the two reagents together had a stronger anti-apoptotic effect than separate usage.
Topics: Animals; Apoptosis; Brain Injuries; Cerebral Hemorrhage; Male; NF-kappa B; Pyrrolidines; Rats; Rats, Wistar; Signal Transduction; Tetradecanoylphorbol Acetate; Thiocarbamates
PubMed: 32729295
DOI: 10.5114/fn.2020.96801 -
Scientific Reports Jul 2022Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on...
Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on protein kinase C activator- and cytokine-mediated cancer cell invasion and migration in MCF-7 and MDA-MB-231 cells and the association between SIRT6 and matrix metalloproteinase-9 (MMP-9) expression. To assess MMP-9 and SIRT6 expression in patients, protein levels in BC tissues were analyzed. MCF-7 and MDA-MB-231 cell viability was analyzed using MTT assays. SIRT6 was silenced in both cell lines and protein secretion, expression, and mRNA levels were analyzed. Transcription factor DNA activity was investigated using luciferase assays. Matrigel invasion assays were used to assess the effects of SIRT6 in both cell lines. SIRT6 and MMP-9 expression in cancer tissues was significantly higher than in paired normal breast tissues. 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) increased MMP-9 expression and cell invasion and migration, but SIRT6 knockdown abolished these effects. SIRT6 overexpression additively increased TPA- and TNF-α-induced MMP-9 expression. SIRT6 knockdown suppressed the mitogen-activated protein kinase (MAPK) signaling pathway and thus TPA- and TNF-α-induced MMP-9 expression. SIRT6 silencing suppressed TPA- and TNF-α-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) expressions in both cell lines, and treatment with MAPK, NF-κB, and AP-1 inhibitors reduced MMP-9 expression. The anti-invasive effects of SIRT6 in BC cells might be mediated by suppression of MAPK phosphorylation and reduction in NF-κB and AP-1 DNA activities, leading to MMP-9 downregulation, suggesting that SIRT6 modulation has the potential to target BC metastasis.
Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; MCF-7 Cells; Matrix Metalloproteinase 9; NF-kappa B; Neoplasm Invasiveness; Sirtuins; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Tumor Necrosis Factor-alpha
PubMed: 35840633
DOI: 10.1038/s41598-022-16405-x -
International Journal of Molecular... Oct 2021The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma...
The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4) or independent (RIPK4) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).
Topics: Animals; Antigens, CD; Apoptosis; Cadherins; Cell Movement; Cell Proliferation; Cells, Cultured; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanocytes; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Protein Kinase C beta; Protein Serine-Threonine Kinases; RNA Interference; RNA, Small Interfering; Transcription Factor RelA; Transplantation, Heterologous
PubMed: 34768934
DOI: 10.3390/ijms222111504 -
PloS One 2022Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and...
Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and inflammation within the gut-liver axis. Studying the hepatic immune response is of high importance as the liver is primarily exposed to gut-derived pathogen-associated molecular patterns. The aim of the present study was to assess the effects of chicken cathelicidin-2 alone or combined with lipoteichoic acid (LTA) or phorbol myristate acetate (PMA) on cell viability, immune response and redox homeostasis in a primary hepatocyte-non-parenchymal cell co-culture of chicken origin. Both concentrations of cathelicidin-2 decreased the cellular metabolic activity and increased the extracellular lactate dehydrogenase (LDH) activity reflecting reduced membrane integrity. Neither LTA nor PMA affected these parameters, and when combined with LTA, cathelicidin-2 could not influence the LDH activity. Cathelicidin-2 had an increasing effect on the concentration of the proinflammatory CXCLi2 and interferon- (IFN-)γ, and on that of the anti-inflammatory IL-10. Meanwhile, macrophage colony stimulating factor (M-CSF), playing a complex role in inflammation, was diminished by the AMP. LTA elevated IFN-γ and decreased M-CSF levels, while PMA only increased the concentration of M-CSF. Both concentrations of cathelicidin-2 increased the H2O2 release of the cells, but the concentration of malondialdehyde as a lipid peroxidation marker was not affected. Our findings give evidence that cathelicidin-2 can also possess anti-inflammatory effects, reflected by the alleviation of the LTA-triggered IFN-γ elevation, and by reducing the M-CSF production induced by PMA. Based on the present results, cathelicidin-2 plays a substantial role in modulating the hepatic immune response with a multifaceted mode of action. It was found to have dose-dependent effects on metabolic activity, membrane integrity, and reactive oxygen species production, indicating that using it in excessively high concentrations can contribute to cell damage. In conclusion, cathelicidin-2 seems to be a promising candidate for future immunomodulating drug development with an attempt to reduce the application of antibiotics.
Topics: Adenosine Monophosphate; Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Chickens; Coculture Techniques; Hepatocytes; Hydrogen Peroxide; Immunity; Inflammation; Interferon-gamma; Interleukin-10; Lactate Dehydrogenases; Liver; Macrophage Colony-Stimulating Factor; Malondialdehyde; Pathogen-Associated Molecular Pattern Molecules; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Cathelicidins
PubMed: 36215285
DOI: 10.1371/journal.pone.0275847 -
Cellular and Molecular Neurobiology Oct 2020Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal...
Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.
Topics: Animals; Cells, Cultured; Central Nervous System; Hippocampus; Neurons; Phorbol Esters; Rats; Receptors, AMPA; Synaptic Transmission; Ubiquitination
PubMed: 32052226
DOI: 10.1007/s10571-020-00809-2