Protein Kinase C Research Articles

Activating PKC-ε induces HIV expression with improved tolerability

Despite suppressive antiretroviral therapy (ART), HIV-1 persists in latent reservoirs that seed new HIV infections if ART is interrupted, necessitating lifelong therapy for people with HIV. Activation of latent HIV during ART could improve recognition and elimination of infected cells by the immune system. Protein kinase C (PKC) isozymes increase HIV transcription and hence are potential latency reversal agents. However, the clinical utility of PKCs for this application is limited due to toxicity, which is poorly understood. Our studies showed that PKC activation with multiple classes of agonists leads to widespread platelet activation, consistent with disseminated intravascular coagulation, at concentrations that were similar to those required for T-cell activation. Differential expression analysis indicated that PKC-ε and PKC-η isoforms are expressed at high levels in human CD4+ T cells but not in platelets. Using structure-based drug design, we developed a novel PKC agonist, C-233, with increased selectivity for PKC-ε. C-233 increased both supernatant HIV RNA and p24 expression ex vivo after treatment of CD4+ T cells from ART-suppressed people with HIV. C-233 was 5-fold more potent for T-cell activation relative to platelet activation. Our studies support the use of structure-based drug design to create selective novel PKC agonists for the safe activation of HIV reservoirs and improved tolerability.

Redox signaling regulation in human spermatozoa: a primary role of peroxiredoxins.

Reactive oxygen species (ROS) play a dual role in mammalian spermatozoa. At high levels, they are detrimental to sperm function since they can promote oxidative stress that produces oxidation of protein, lipids, and sperm DNA. This oxidative damage is associated with male infertility. On the other hand, when ROS are produced at low levels, they participate in the redox signaling necessary for sperm capacitation. Capacitation-associated ROS are produced by the sperm oxidase, whose identity is still elusive, located in the plasma membrane of the spermatozoon. ROS, such as superoxide anion, hydrogen peroxide, nitric oxide, and peroxynitrite, activate protein kinases and inactivate protein phosphatases with the net increase of specific phosphorylation events. Peroxiredoxins (PRDXs), antioxidant enzymes that fight against oxidative stress, regulate redox signaling during capacitation. Among them, PRDX6, which possesses peroxidase and calcium-independent phospholipase A2 (iPLA2) activities, is the primary regulator of redox signaling and the antioxidant response in human spermatozoa. The lysophosphatidic acid signaling is essential to maintain sperm viability by activating the phosphatidylinositol 3-kinase/protein kinase (PI3K/AKT) pathway, and it is regulated by PRDX6 iPLA2, protein kinase C (PKC), and receptor-type protein tyrosine kinase. The understanding of redox signaling is crucial to pave the way for novel diagnostic tools and treatments of male infertility.

Phosphorylation of distal C-terminal residues promotes TRPV4 channel activation in response to arachidonic acid.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable channel activated by diverse physical and chemical stimuli, including mechanical stress and endogenous lipid arachidonic acid (AA) and its metabolites. Phosphorylation of TRPV4 by protein kinase A (PKA) and protein kinase C (PKC) is a predominant mechanism for channel regulation, especially in the cytoplasmic domains due to their importance in protein assembly, and channelopathies. However, studies corresponding to phosphorylation sites for these kinases remain incomplete. We investigated the role of Ser-823 residue as a potential phosphorylation site in regulating TRPV4 activity and chemical agonist-induced channel activation. Using mass spectrometry, we identified a new phosphorylation site Ser-823 residue and confirmed the previously known phosphorylation site Ser-824 in the C-terminal tail. The low level of phosphorylation at Ser-823 was stimulated by PKC and to a lesser extent by PKA in human coronary artery endothelial cells (HCAECs) and human embryonic kidney 293 (HEK 293) cells. AA-induced TRPV4 activation was enhanced in the phosphomimetic S823E but was blunted in the S823A/S824A mutants, whereas the channel activation by the synthetic agonist GSK1016790A was unaffected. Further, TRPV4 activation by AA but not GSK1016790A was blunted or abolished by PKA inhibitor alone or in combination with PKC inhibitor, respectively. Using computational modeling, we refined a previously proposed structural model for TRPV4 regulation by Ser-823 and Ser-824 phosphorylation. Together, these results provide insight into how stimuli-specific TRPV4 activation is regulated by the phosphorylation of discrete residues (e.g., Ser-823 and Ser-824) in the C-terminal domains of the TRPV4 channel.

Increased phosphorylation of AMPKα1 S485 in colorectal cancer and identification of PKCα as a responsible kinase.

The present study attempts to examine the biological effect of phosphorylation of AMPKα1 S485 and identify the responsible kinase in colon cancer cells. Thus, our results showed that S485 phosphorylation was increased in colorectal cancer specimens as compared with adjacent normal tissues, which was inversely correlated to phosphorylation of T172. Our study further revealed that phosphorylation of S485 on AMPKα1 plays a promoting role in cell proliferation, colony formation, migration and growth of Xenograft tumor. Furthermore, we identified PKCα as a kinase specific for phosphorylation of S485. First, under the basal condition, S485 phosphorylation was blunted by Gö6983, a pan PKC inhibitor, but not by Akt inhibitor, MK2206, although the latter countered off the insulin-stimulated phosphorylation. Second, the phosphorylation was enhanced by PMA and attenuated by sgRNA for PKCα, but not by PKCγ and PKCδ, neither by siRNA for Akt1. Third, the phosphorylation was suppressed by shRNA for PLCγ1. Fourth, the phosphorylation was enhanced by ectopically expressing a constitutively active mutant of PKCα, but not PKCγ. Finally, the increase of S485 phosphorylation by high glucose or palmitic acid was almost completely abolished by Gö6983. Altogether, our data reinforced the tumor suppressive function of AMPK and demonstrated that PKCα is a major kinase responsible for phosphorylation of S485, which contributes to one of the mechanisms underlying the regulation of AMPK in cancer cells in response to nutritional conditions.

Antiplatelet Effects of DMPC-Based Synthetic High-Density Lipoproteins: Exploring Particle Structure and Noncholesterol Efflux Mechanisms.

Platelet activation is a key factor in the development of cardiovascular diseases. High-density lipoprotein (HDL) is known for its cardioprotective activities including antithrombotic actions. While HDL mimetics have been explored for their potential to regulate thrombosis, their influence on platelet activity remains unclear. This study explores the capacity of synthetic HDL (sHDL) to modulate platelet function and investigates the underlying mechanisms. We examined the effects of sHDL, formulated with various ApoA1 mimetic peptides (18A, 5A, and 22A) and full-length ApoA1 protein, all complexed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), on platelet function. DMPC-based sHDL demonstrated pronounced antiplatelet effects across all formulations. Comparison with DMPC micelles showed that all sHDL molecules were more effective, highlighting the crucial role of the protein-phospholipid complex in reducing platelet reactivity. Further analysis revealed that DMPC sHDL dose-dependently inhibited various platelet functions, including aggregation, integrin activation, α-granule secretion, protein kinase C (PKC) activation, and platelet spreading. Mechanistic studies demonstrated that DMPC sHDL's antiplatelet effects are not entirely dependent on cholesterol efflux, despite effectively reducing total platelet cholesterol. Furthermore, sHDL's activity was found to be independent of scavenger receptor BI (SR-BI). Notably, inhibition of the CD36 receptor markedly attenuated sHDL's antiplatelet activity and uptake, suggesting a novel mechanism distinct from that of native HDL. In summary, DMPC sHDL modulates platelet function through a synergistic action between protein and phospholipid components, primarily via CD36 receptor engagement. These insights pave the way for novel antiplatelet therapies utilizing sHDL's distinct properties.

Enhanced vascular contraction induced by exposure to angiotensin II mediated by endothelin-1 biosynthesis following PKCβ activation.

Protein kinase C (PKC) reportedly plays a role in the pathogenesis of many vascular dysfunction-related conditions. In this study, we investigated whether PKCβ is associated with vascular contractile changes induced by angiotensin II (Ang II) exposure. Long-term (24 h) treatment of rat aortae and mesenteric arteries in Ang II-containing culture medium enhanced 5-hydroxytrypatamine (5-HT)-induced vascular contraction in a dose-dependent manner, in association with enhanced phosphorylation of PKCβ S660. Increased contraction induced by Ang II treatment was also observed in endothelium-denuded aorta. Enhanced contraction induced by Ang II was markedly diminished by knockout of the PKCβ gene or treatment with LY333531 and CGP53353 (PKCβ inhibitors). Cycloheximide (protein synthesis inhibitor) blocked the Ang II-induced enhanced contraction. Gene expression profiling and real-time PCR analyses showed marked upregulation of endothelin-1 (ET-1) expression in Ang II-treated aorta, but was not observed in PKCβ-knockout aorta. Ang II increased the secretion of ET-1 peptide in endothelium-intact and -denuded aortae. Ang II-induced enhancement of vascular contraction was diminished by BQ-123 (ETAR blocker). In vivo treatment with Ang II (250 ng/kg/min) for 7 days increased phosphorylation of PKCβ S660 in rat vascular tissue and increased the in vitro contractile responses to 5-HT and in vivo systolic blood pressure, but these changes were largely absent in PKCβ-knockout experiments. These data suggest that long-term exposure to Ang II increases vascular smooth muscle contraction and blood pressure elevation, mediated by activation of PKCβ and subsequent biosynthesis of ET-1 in vascular smooth muscle cells.

Exploration of Genetic Variants Associated with Lung Cancer: A Comprehensive Review

Lung cancer (LC) is a widespread and prevalent disease with a high death rate. The occurrence of this terrible disease is greatly influenced by genetic, occupational and environmental risk factors. Therefore, the present review aimed to investigate and accumulate the information regarding the causative gene of lung cancer. The articles included in this study were retrieved from various online sources, including Cochrane Library, Google Scholar, EMBASE, PubMed and Web of Science databases up to October 2024. Data were extracted based on the PRISMA 2020 guidelines. The study recognized several genetic mutations that are accountable for the progression of lung cancer. The most common identified lung cancer associated with genes include XRCC1, XRCC4, KRAS, TP53, EGFR, PI3KCA, ERCC2/XPD MET, ALK1, BRAF, HER2, LTKB-1/STK11, AKT1 and MAP2K1. Additionally, HER2, MAP2K1/MEK1, AKT, NRAS and various combinations of co-occurring mutations were also noted as stimulators of this cancer. These genes instigate lung cancer either by impairing DNA repair efficiency or interacting with various signaling pathways that result in cell proliferation, differentiation, angiogenesis, invasion and metastasis, such as, PI3k/AKT (phosphatidylinositol 3-kinase/protein kinase B) pathway, MAPKs (mitogen-activated protein) pathway, PKC (protein kinase C), JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway, as well as RAS signaling. Studying epigenetic mechanisms in lung cancer is crucial to understand the genetic variant linked to the disease and identify potential therapeutic targets and molecular targets of drugs. Bangladesh Pharmaceutical Journal 28(1): 114-125, 2025 (January)

Validation of Machine Learning-assisted Screening of PKC Ligands: PKC Binding Affinity and Activation.

Protein kinase C (PKC) is a family of serine/threonine kinases, and PKC ligands have the potential to be therapeutic seeds for cancer, Alzheimer's disease, and human immunodeficiency virus infection. However, in addition to desired therapeutic effects, most PKC ligands also exhibit undesirable pro-inflammatory effects. The discovery of new scaffolds for PKC ligands is important for developing less inflammatory PKC ligands, such as bryostatins. We previously reported that machine learning combined with our knowledge of the pharmacophore yielded 15 PKC ligand candidates, but we did not evaluate their PKC binding affinities fully. In this paper, PKC binding affinities of four candidates were examined to assess their potential as PKC ligands and to validate machine learning-assisted screening. Although compound 3' did not bind to PKC C1 domains, 1a, 2', and 4a exhibited moderate PKC binding affinities, suggesting that machine learning-assisted screening is advantageous in identifying new PKC ligand scaffolds.

Synthesis and preclinical evaluation of tigilanol tiglate analogs as latency-reversing agents for the eradication of HIV.

Tigilanol tiglate (EBC-46) is a selective modulator of protein kinase C (PKC) isoforms that is Food and Drug Administration (FDA) approved for the treatment of mast cell tumors in canines with up to an 88% cure rate. Recently, it has been FDA approved for the treatment of soft tissue sarcomas in humans. The role of EBC-46 and, especially, its analogs in efforts to eradicate HIV, treat neurological and cardiovascular disorders, or enhance antigen density in antigen-targeted chimeric antigen receptor-T cell and chimeric antigen receptor-natural killer cell immunotherapies has not been reported. Enabled by our previously reported scalable synthesis of EBC-46, we report herein the systematic design, synthesis, and evaluation of EBC-46 analogs, including those inaccessible from the natural source and their PKC affinities, ability to translocate PKC, nuclear factor κB activity, and efficacy in reversing HIV latency in Jurkat-Latency cells. Leading analogs show exceptional PKC affinities, isoform selectivities, and functional activities, serving as promising candidates for therapeutic applications.

Hyperglycemia-induced oxidative stress in the development of diabetic retinopathy

Diabetic retinopathy (DR), the leading cause of preventable blindness, is primarily caused by chronic hyperglycemia-induced oxidative stress. This review examines molecular mechanisms by which hyperglycemia-induced oxidative damage transmits into retinal tissue. We summarize the major pathways: polyol activation, advanced glycation end-products (AGEs), protein kinase C (PKC) dysregulation, and hexosamine pathway. Oxidative stress leads to mitochondrial dysfunction, apoptosis of retinal endothelium and pigment epithelium and activation of cytokines, as well as overexpression of vascular endothelial growth factor (VEGF). These processes together cause vascular leakage, macular edema and pathological angiogenesis. Trials of oxidative stress therapies (e.g. antioxidants, PKC inhibitors e.g., ruboxistaurin, and anti-VEGF agents e.g., ranibizumab, bevacizumab, aflibercept) as well as mitochondria targeted therapies are considered potential therapeutic approaches to improve DR prognosis. Further studies on DR pathophysiology and treatment are recommended to develop effective interventions for this vision threatening condition. Better prevention and management of DR requires early intervention and biomarker-based approaches.

PCSK9 Expression in Vascular Smooth Muscle Cells: Role of Insulin Resistance and High Glucose

Beyond the regulation of cholesterol metabolism, a number of extrahepatic functions of proprotein convertase subtilisin/kexin type 9 (PCSK9) have been increasingly identified. The main purpose of this study was to verify whether PCSK9 expression in vascular smooth muscle cells (VSMC) is influenced by insulin resistance and high glucose (HG). In cultured rat aortic VSMC from lean insulin-sensitive Zucker rats (LZRs) and obese insulin-resistant Zucker rats (OZRs), a classical animal model of insulin resistance, we evaluated PCSK9 expression with or without the monoclonal antibodies against PCSK9 Alirocumab and Evolocumab or the synthetic PCSK9-binding peptide PEP 2-8. Effects and molecular mechanisms underlying altered PCSK9 expression were evaluated by proliferation and migration assay, reactive oxygen species (ROS) production, and involvement of PKC, NADPH-oxidase, MAPK/ERK-1/2 pathway activation. As a result, we found that, in comparison with LZR, VSMC from OZR showed basal PCSK9 overexpression mitigated by Alirocumab, Evolocumab, PEP 2-8, and the inhibitors of PKC, NADPH-oxidase, and MAPK. The finding of PCSK9 upregulation in VSMC from OZR paralleled with increased ROS production, proliferation, and migration. HG increased PCSK9 expression in VSMC from LZR, but not in OZR, via oxidative stress and with effects reduced by PCSK9 inhibitors. These findings suggest that a dysregulation of PCSK9 in VSMC could be involved in vascular damage in metabolic disorders, such as obesity and diabetes.

Alpha 1-antitrypsin mitigates salt-sensitive hypertension in juvenile mice by reducing diacylglycerol concentrations and protein kinase C activity in kidney membranes.

Recombinant alpha-1 antitrypsin (AAT) therapy has been shown to have beneficial effects to mitigate the progression of various diseases. Here, we hypothesized that administration of pharmaceutical-grade human AAT (hAAT) is effective in mitigating hypertension induced by salt-loading in juvenile mice by reducing the concentration of diacylglycerols (DAGs) and activity of protein kinase C (PKC) in the kidney. Four-week old 129Sv mice were salt-loaded to induce hypertension and then administered hAAT or vehicle. Administration of hAAT was found to significantly reduce high blood pressure in both the active and inactive cycles of the 129Sv hypertensive mice. A lipidomic analysis showed decreased concentrations of multiple diacylglycerols in kidney cortex membrane fractions from mice treated with hAAT compared to vehicle. PKC activity was less in the 129Sv mice that received hAAT compared to vehicle. Western blotting and immunohistochemistry analysis showed the density of the sodium-potassium-chloride co-transporter (NKCC2) was significantly reduced in kidney cortex membrane fractions of juvenile mice that received hAAT compared to vehicle. Taken together, this study demonstrates a new protective effect of hAAT in normalizing blood pressure after the development of saltinduced hypertension in juvenile mice in a mechanism involving a decrease in NKCC2 membrane expression, presumably due to decreased levels of DAGs in the plasma membrane and a subsequent decrease in PKC activity.

The protein kinase C modulator bryostatin-1 therapeutically targets microglia to attenuate neuroinflammation and promote remyelination.

In multiple sclerosis (MS), microglia and macrophages within the central nervous system (CNS) play an important role in determining the balance among demyelination, neurodegeneration, and myelin repair. Phagocytic and regenerative functions of these CNS innate immune cells support remyelination, whereas chronic and maladaptive inflammatory activation promotes lesion expansion and disability, particularly in the progressive forms of MS. No currently approved drugs convincingly target microglia and macrophages within the CNS, contributing to the lack of therapies aimed at promoting remyelination and slowing disease progression for individuals with MS. Here, we found that the protein kinase C (PKC)-modulating drug bryostatin-1 (bryo-1), a CNS-penetrant compound with an established human safety profile, shifts the transcriptional programs of microglia and CNS-associated macrophages from a proinflammatory phenotype to a regenerative phenotype in vitro and in vivo. Treatment of microglia with bryo-1 stimulated scavenger pathways, phagocytosis, and secretion of factors that prevented the activation of neuroinflammatory reactive astrocytes while also promoting neuroaxonal health and oligodendrocyte differentiation. In line with these findings, systemic treatment of mice with bryo-1 augmented remyelination after a focal demyelinating injury. Our results demonstrate the potential of bryo-1 and possibly a wider class of PKC modulators as myelin-regenerative and supportive agents in MS and other neurologic diseases.

Activation of protein kinase C decreases equilibrative nucleobase transporter 1-mediated substrate uptake via phosphorylation of threonine 231.

Protein kinase C (PKC) signalling has been shown to be dysregulated in various cancers including acute lymphoblastic leukemia (ALL). We have previously determined that changes in the expression levels of SLC43A3-encoded equilibrative nucleobase transporter 1 (ENBT1) can significantly alter 6-mercaptopurine (6-MP) toxicity in ALL cells. 6-MP is a common drug used in ALL chemotherapy. Furthermore, it has been reported that activation of PKC by phorbol 12-myristate 13-acetate (PMA) impacts nucleobase uptake via an ENBT1-like transporter in Lilly Laboratories Culture-Porcine Kidney 1 (LLC-PK1) cells. We hypothesized that activation of PKC would also alter ENBT1-mediated uptake of nucleobases in leukemia cell models. Using MOLT-4, SUP-B15, and K562 cells, we incubated the cells with PMA or its inactive isoform 4α-PMA for 30min and determined changes to ENBT1-mediated substrate uptake. All of the cell lines tested showed decreased ENBT1-mediated substrate uptake when exposed PMA, relative to that observed using 4α-PMA. Pre-incubation with the broad-spectrum PKC inhibitor, Gö6983, reversed the decrease caused by PMA. Finally, to determine the residue responsible for this PKC-mediated effect, we transiently transfected HEK293 cells (which do not express endogenous ENBT1) with wild-type SLC43A3 transcript or constructs mutated to modify the predicted PKC sites in ENBT1. We found that the mutation of threonine 231 to alanine prevents the decrease in ENBT1-mediated uptake following incubation with PMA, suggesting its involvement. This study shows that activation of PKC decreases ENBT1-mediated uptake, suggesting that aberrant activation of PKC in ALL could decrease ENBT1-mediated 6-MP uptake potentially leading to decreased therapeutic efficacy.

Panretinal photocoagulation for the treatment of proliferative diabetic retinopathy

After the completion of DRS study in 1979, Panretinal Photocoagulation (PRP) became the gold standard for the treatment of proliferative diabetic retinopathy.Light from the laser is absorbed by the retinal pigment epithelium (RPE), and the underlying choroid and it is converted to thermal energy. Those thermal burns denature tissue protein, causing local retinal cell death, coagulative necrosis and eventually scarring. In that respect, laser photocoagulation leads to diminished oxygen demands of the areas of capillary non perfusion, and reduced expression of vasoactive factors such as vascular endothelial growth factor (VEGF) and protein kinase C (PKC) in the retina. As a result, adequate oxygen and nutrients are diverted towards the non‐ischemic retinal areas.As documented by the DRS study, after a 5‐year follow‐up period, the rates of severe visual loss were diminished by at least 50% in all eyes treated with PRP in comparison with untreated control eyes. The greatest beneficial effect was shown for treated eyes with high risk PDR characteristics (any NVD with extent greater than or equal to one‐fourth disc area without vitreous or preretinal hemorrhage, any NVD with vitreous or preretinal hemorrhage,any NVE with extent greater than or equal to one‐half disc area, with vitreous or preretinal hemorrhage). Harmful effects of PRP includes peripheral visual field loss, decrease in visual acuity, nyctalopia etc. Treatment of non‐high risk PDR cases, could also be proposed under certain circumstances such as in patients with difficulty to attend regular appointments, advanced diabetic disease in the fellow eye etc.Although anti‐VEGF agents have been approved for the treatment of PDR, PRP remains an effective therapeutic option and the choice of treatment could be made on an individualized basis. However, in high risk PDR patients with co‐existing maculopathy the addition of anti‐VEGF injections to PRP treatment is advisable.

Spatiotemporal distribution of PKCα, PIP3, Moesin, Cdc42, MARCKS, Scriblle, and Arf6 before directed cell migration in monolayers.

Protein kinase Cα (PKCα) has an important role in directed cell migration. After a mechanical wounding, PKCα rapidly accumulates at cell edges adjacent to the wounded cell and regulates cell migration. However, the proteins downstream of PKCα that mediate directed signaling remain unknown. In this study, we examined the spatiotemporal dynamics of PKCα, PIP3, Moesin, Cdc42, MARCKS, Scribble, and Arf6 before directed migration. After wounding, PIP3, Moesin, and Cdc42 accumulated at the cell edge near the wounded cells later than PKCα. In contrast, MARCKS moved away from the plasma membrane without polarization, and Scribble and Arf6 exhibited no significant translocation. The inhibition of PIP3 suppressed the accumulation of Moesin and Cdc42, suggesting that PIP3 regulates Moesin and Cdc42. In particular, the inhibition of PKCα completely inhibited the translocation of all factors, indicating that PKCα is a central regulator in early signaling after wounding and before directional migration.

Phosphorylation of (Ser 291) in the linker insert of Syk negatively regulates ITAM signaling in platelets

Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl3 injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling in vitro but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses in vivo.

P62 Binding to Protein Kinase C Regulates HIV-1 gp120 V3 Loop Induced Microglial Inflammation.

The main pathogenic mechanism of HIV-associated neurocognitive disorders (HAND) is neuronal apoptosis induced by inflammatory mediators, in which microglial inflammation plays a crucial role. However, the exact pathogenic mechanism remains unclear. Previous studies have shown that the HIV-1 gp120 V3 loop can trigger inflammation in CHME-5 microglia. p62 is a post-translational modified multidomain protein that is involved in the regulation of autophagy and is closely related to neuroinflammation. In this study, we found that p62 knockout down-regulated the expression of MCP-1, IL-6 and COX-2, and improved the inflammation of HIV-1 gp120 V3 loop induced microglia, while overexpression of p62 up-regulated the expression of MCP-1, IL-6 and COX-2, and promoted the inflammation of microglia. In addition, protein kinase C (PKC) knockout down-regulated the expression of MCP-1, IL-6 and COX-2 and inhibited the activation of IKK/ NF-κ B pathway, while tumor necrosis factor receptor-associated factor 6 (TRAF6) knockout had no significant effect on the expression of MCP-1, IL-6 and COX-2. Co-immunoprecipitation showed that p62 was bound and interacted with PKC. Inhibition of IKK/ NF-κ B pathway can down-regulate the expression of MCP-1, IL-6 and COX-2, and improve the inflammatory response of microglia. Our research further found that inhibition of IKK/ NF-κ B can decrease the expression of Caspase-3 and reduce the apoptosis of neurons in the co-culture of CHME-5 microglia and primary mouse neurons. The results of this study suggest that HIV-1 gp120 V3 loop induced CHME-5 microglial inflammation may be activated by the direct binding of p62 and PKC through the IKK/ NF-κ B signaling pathway, and these findings provide an important reference for the prevention and treatment of HAND.

Inflammatory Stimulation Upregulates the Receptor Transporter Protein 4 (RTP4) in SIM-A9 Microglial Cells

The receptor transporter protein 4 (RTP4) is a receptor chaperone protein that targets class A G-protein coupled receptor (GPCR)s. Recently, it has been found to play a role in peripheral inflammatory regulation, as one of the interferon-stimulated genes (ISGs). However, the detailed role of RTP4 in response to inflammatory stress in the central nervous system has not yet been fully understood. While we have previously examined the role of RTP4 in the brain, particularly in neuronal cells, this study focuses on its role in microglial cells, immunoreactive cells in the brain that are involved in inflammation. For this, we examined the changes in the RTP4 levels in the microglial cells after exposure to inflammatory stress. We found that lipopolysaccharide (LPS) treatment (0.1~1 µg/mL, 24 h) significantly upregulated the RTP4 mRNA levels in the microglial cell line, SIM-A9. Furthermore, the interferon (IFN)-β mRNA levels and extracellular levels of IFN-β were also increased by LPS treatment. This upregulation was reversed by treatment with neutralizing antibodies targeting either the interferon receptor (IFNR) or toll-like receptor 4 (TLR4), and with a TLR4 selective inhibitor, or a Janus kinase (JAK) inhibitor. On the other hand, the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, significantly enhanced the increase in RTP4 mRNA following LPS treatment, whereas the PKC inhibitor, calphostin C, had no effect. These findings suggest that in microglial cells, LPS-induced inflammatory stress activates TLR4, leading to the production of type I IFN, the activation of IFN receptor and JAK, and finally, the induction of RTP4 gene expression. Based on these results, we speculate that RTP4 functions as an inflammation-responsive molecule in the brain. However, further research is needed to fully understand its role.

Marcks overexpression in retinal ganglion cells promotes optic nerve regeneration

Regeneration of injured central nervous system (CNS) axons is highly restricted, leading to permanent neurological deficits. The myristoylated alanine-rich C-kinase substrate (MARCKS) is a membrane-associated protein kinase C (PKC) substrate ubiquitously expressed in eukaryotic cells, plays critical roles in development, brain plasticity, and tissues regeneration. However, little is known about the role of Marcks in CNS axon regeneration. Here we show that Marcks overexpression promotes robust axon regeneration either before or after optic nerve crush, but insignificantly impacts neuronal survival. Notably, immunostaining and RNA sequencing demonstrate that Marcks overexpression does not affect known regeneration-associated genes and pathways. Furthermore, combining CNTF which activates the JAK-STAT3 pathway and Marcks overexpression further enhances axon regeneration. Finally, we demonstrate functionally essential effector domain (ED) of MARCKS has similar effects on inducing axon regeneration in RGCs. These results suggest that manipulating Marcks and its ED may become a therapeutic approach to promote axon regeneration after CNS injury.