-
Neurotoxicology Dec 2022Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level...
Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level of GAP-43 in the striatum of rats that receives rich dopaminergic terminals. Using PC12 cells as dopaminergic neuronal models, we further found that AMPH inhibited GAP-43 and GAP-43 phosphorylation in PC12 cells. The reduced GAP-43 was correlated with neurite injury of PC12 cells. The PKCβ1, an upstream molecule of GAP-43, was also inhibited by AMPH. Phorbol 12-myristate 13-acetate (PMA) as a specific activator of PKC increased levels of PKCβ1 and GAP-43, and efficiently prevented neurite degeneration of PC12 cells induced by AMPH. On the other side, enzastuarin, an inhibitor of PKC, decreased levels of PKCβ1 and GAP-43, and caused neurite injury of PC12 cells. Together, our results suggest that AMPH induces neurite injury in PC12 cells through inhibiting PKCβ1/GAP-43 pathway.
Topics: Animals; Rats; Amphetamine; PC12 Cells; Neurites; GAP-43 Protein; Tetradecanoylphorbol Acetate; Dopamine
PubMed: 36150536
DOI: 10.1016/j.neuro.2022.09.004 -
IScience Nov 2021Thrombocytopenia-absent radius (TAR) syndrome is caused by RBM8A insufficiency. We generated megakaryocyte-specific knockout (KO) mice that exhibited marked...
Thrombocytopenia-absent radius (TAR) syndrome is caused by RBM8A insufficiency. We generated megakaryocyte-specific knockout (KO) mice that exhibited marked thrombocytopenia, internal hemorrhage, and splenomegaly, providing evidence that genetic deficiency of causes a disorder of platelet production. KO mice accumulated low-ploidy immature megakaryocytes in the bone marrow and exhibited defective platelet activation and aggregation. Accordingly, depletion of Y14 (RBM8A) in human erythroleukemia (HEL) cells compromised phorbol-ester-induced polyploidization. Notably, Y14/RBM8A deficiency induced both p53 and p21 in megakaryocytes and HEL cells. Treatment with a p53 inhibitor restored differentiation of KO megakaryocytes and unexpectedly activated Y14 expression in HEL cells knockout partially restored megakaryocyte differentiation by reversing cell-cycle arrest and increased platelet counts of KO, indicating that excess p53 in part accounts for thrombocytopenia in TAR syndrome. This study provides evidence for the role of the Y14-p53 circuit in platelet production and a potential therapeutic strategy.
PubMed: 34816104
DOI: 10.1016/j.isci.2021.103368 -
The Journal of Investigative Dermatology Sep 2021Epidermal keratinocytes (KCs) rapidly proliferate to repair the skin barrier, and a strict control of division is necessary for healthy tissue homeostasis. However, the...
Epidermal keratinocytes (KCs) rapidly proliferate to repair the skin barrier, and a strict control of division is necessary for healthy tissue homeostasis. However, the pathways that restrain proliferation after epidermal stress are not known. AMPK is an important signaling mediator of energy metabolism previously associated with skin stress and cancer; yet, its explicit impact on KC growth is not known. To examine the requirement of epidermal AMPK in physiologic skin repair, we genetically deleted AMPK within all adult, keratin 14‒expressing KCs of mice. AMPK loss resulted in hyperproliferation and hyperactive mTOR signaling after acute wounding, UVB exposure, and phorbol ester application. This excessive division could be completely blocked by the mTORC1 inhibitor rapamycin. Moreover, we establish that the diabetes drug metformin depends on AMPK to suppress stress-induced KC proliferation. Collectively, these findings show that KC AMPK restrains mTORC1 to control epidermal proliferation after tissue injury.
Topics: Adenylate Kinase; Animals; Cell Proliferation; Cells, Cultured; Energy Metabolism; Keratin-14; Keratinocytes; Mechanistic Target of Rapamycin Complex 1; Metformin; Mice; Mice, Transgenic; Sirolimus; Skin; Stress, Physiological; Ultraviolet Rays
PubMed: 33741392
DOI: 10.1016/j.jid.2020.12.036 -
The Journal of Biological Chemistry Jan 2020β1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the gene, which also encodes the β2- and β3-chimaerin isoforms....
β1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the gene, which also encodes the β2- and β3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of β1-chimaerin, β1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that β1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, β1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that β1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in β1-chimaerin that is absent in β1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that β1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.
Topics: Alternative Splicing; Amino Acid Motifs; Animals; COS Cells; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Chimerin Proteins; Chlorocebus aethiops; Diglycerides; Exons; Gene Silencing; Humans; Protein Domains; Protein Isoforms; RNA, Small Interfering; Sequence Deletion; rac1 GTP-Binding Protein
PubMed: 31871052
DOI: 10.1074/jbc.RA119.008688 -
Scientific Reports May 2021Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation...
Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.
Topics: Actin Cytoskeleton; Humans; Organelles; Platelet Activation; Tetradecanoylphorbol Acetate
PubMed: 34006947
DOI: 10.1038/s41598-021-89799-9 -
PloS One 2020Intense dance training leads to inflammation, which may impair the health and performance of the practitioners. Herein, we evaluate the effect of a single street dancing...
Intense dance training leads to inflammation, which may impair the health and performance of the practitioners. Herein, we evaluate the effect of a single street dancing class on the profile of muscle enzymes, lymphocyte activation, and cell surface CD62L expression. We also investigated the correlation between muscle enzymes, adhesion molecules, and lymphocyte activation in dancers. Fifteen male participants (mean ± standard error: age 22.4 ± 1.08 years, body mass index 24.8 ± 0.69 kg/m2, body fat 12.3 ± 1.52%), who were amateur dancers, had blood samples collected previously and subsequent to a high-intensity street dance class. After the class, dancers showed an increase in total lymphocyte count (2.0-fold), creatine kinase (CK)-NAC (4.87%), and CK-MB (3.36%). We also observed a decrease (2.5-fold) in reactive oxygen species (ROS) produced by lymphocytes, under phorbol myristate acetate-stimulated environments. Following the dance class, CD62L expression in lymphocytes decreased (51.42%), while there was a negative correlation between the intensity of the exercise and CD62L expression (r = -0.73; p = 0.01). Lymphocytes were less responsive to stimuli after a single bout of street dancing, indicating transient immunosuppression.
Topics: Creatine Kinase, MB Form; Dancing; Heart Rate; Humans; Inflammation; L-Selectin; Lymphocyte Activation; Lymphocyte Count; Male; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Young Adult
PubMed: 32956398
DOI: 10.1371/journal.pone.0239516 -
Frontiers in Immunology 2022Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune...
Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.
Topics: Animals; Antibodies, Monoclonal; Cattle; Cross Reactions; Dolphins; Enzyme-Linked Immunosorbent Assay; Goats; Interferon-gamma; Interleukin-17; Ionomycin; Leukocytes, Mononuclear; Recombinant Proteins; Swine; T-Lymphocytes; Tetradecanoylphorbol Acetate; Zebrafish
PubMed: 35185884
DOI: 10.3389/fimmu.2022.786396 -
Biochemical and Biophysical Research... Apr 2023Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination...
Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.
Topics: Phosphorylation; Protein Kinase C; Signal Transduction; Myoblasts, Skeletal
PubMed: 36773343
DOI: 10.1016/j.bbrc.2023.02.010 -
Frontiers in Immunology 2022The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ) show reduced surface...
The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δγγ (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.
Topics: CD3 Complex; Humans; Interleukin-2; Leucine; Phorbol Esters; Receptors, Antigen, T-Cell; Tumor Necrosis Factor-alpha
PubMed: 36119034
DOI: 10.3389/fimmu.2022.978658 -
The Journal of Neuroscience : the... Sep 2021Post-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...
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. 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.
Topics: Amino Acid Substitution; Animals; Female; Gene Knock-In Techniques; Hippocampus; Male; Mice; Mice, Knockout; Miniature Postsynaptic Potentials; Munc18 Proteins; Mutation, Missense; Neuronal Plasticity; Phorbol Esters; Phosphorylation; Point Mutation; Protein Kinase C; Protein Processing, Post-Translational; Purkinje Cells; Recombinant Proteins; Synaptic Transmission
PubMed: 34290081
DOI: 10.1523/JNEUROSCI.0347-21.2021