S100 protein expression in PKC-fused blue naevi, cellular blue naevi and PRKAR1A-inactivated pigmented epithelioid melanocytomas.

Recent data have redefined the genetic spectrum of pigmented epithelioid melanocytomas (PEMs). PEM is now defined by a secondary genetic event, a protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A) inactivation, that confers the specific cytomorphology of the entity, but this event can arise within a naevus with a genetic background of common, blue or Spitz type. PKC-fused melanocytic proliferations, although they can exhibit PEM-like morphological features, have now been regrouped within the blue group of tumours. Past studies have shown that the latter group tends to lose S100 expression. Herein, we studied the nuclear expression of S100 by immunohistochemistry in 73 PKC-fused benign blue naevi. Histologically, the most frequent pattern found in PKC-fused blue naevi (51%) was a dermal biphasic architecture associated with a horizontal band of medium-sized bland melanocytes in the upper papillary dermis, with a deeper dermal expansion combining spindled and dendritic melanocytes with occasional small nests of bland melanocytes within a fibrous background. A PEM-like hyperpigmented hyperplasia of the epidermis was seen in 32% of cases. The immunohistochemical study found in 31 of the 37 (84%) dermal biphasic PKC-fused melanocytic tumour cases a significant loss of nuclear expression of S100 (in more than 50% of cells) in the superficial horizontal dermal band area and in 68% of the biphasic dermal component. However, the hyperpigmented PEM-like junctional components were not assessable by immunohistochemistry. An exploratory analysis of S100 expression in 21 blue naevi and in 25 PEM with confirmed PRKAR1A inactivation was also performed. In blue naevi, a loss of nuclear S100 expression in more than 50% of melanocytes was found in over 70% of these lesions both in the dendritic and epithelioid dermal components. By contrast, nuclear expression of S100 was most often preserved in PEM with PRKAR1A inactivation (85% preservation in the epithelioid component). These results suggest that searching for S100 expression loss by immunohistochemistry may be helpful in the diagnosis of PKC-fused blue naevus similarly as in dendritic and cellular blue naevi. This simple test, especially if a band-like structure is present in the upper dermis, can effectively support this diagnosis, as a genetic confirmation of these benign tumours is not warranted.

Differential activation of six galanin receptors by the spexin peptide in yellowtail kingfish (Seriola lalandi).

Spexin (SPX1) is a novel neuropeptide composed of 14 amino acids and well conserved across vertebrates, and it has been implicated in various physiological functions via galanin receptor 2 (GALR2) and GALR3. However, the detailed signaling pathways mediating its actions in target cells are still largely unknown. Accordingly, we addressed this issue in the present study using yellowtail kingfish as a model. SPX1 significantly increased CRE-luc activity in COS-7 cells expressing its cognate receptors GALR2a and GALR2b, and this stimulatory effect was attenuated by two inhibitors of the PKA pathway. Similarly, an evident induction of SRE-luc activity was observed when COS-7 cells transfected with GALR1b, GALR2a, GALR2b, GALR type 1, or GALR type 2 were challenged with SPX1, and two blockers of the PKC pathway suppressed this stimulatory action. Moreover, SPX1 markedly elevated NFAT-RE-luc activity in COS-7 cells expressing GALR1a, GALR2a, or GALR2b, and this promotion was inhibited by two antagonists of the Ca2+ route. Overall, our results have revealed that activation of six yellowtail kingfish galanin receptors by the SPX1 peptide may occur with different downstream signaling events, which could account for its pleotropic functions.

Discovery of Kuraridin as a Potential Natural Anti-Melanogenic Agent: Focusing on Specific Target Genes and Multidirectional Signaling Pathways.

Abnormal melanogenesis upon UV exposure causes excessive oxidative stress, leading to hyperpigmentation disorders. As a key rate-limiting enzyme in melanogenesis, tyrosinase is considered a primary target for depigmenting agents. Sophora flavescens is used as a food and in traditional medicine as a valuable source of prenylated flavonoids. The present study aimed to elucidate the anti-melanogenic effect and potential mechanism of kuraridin, one of the major prenylated flavonoids. Kuraridin showed anti-tyrosinase activity with an IC50 value in the nanomolar range, superior to that of kojic acid, a positive control. It significantly reduced tyrosinase activity with the least cytotoxicity, suppressing melanogenesis in α-MSH-induced B16F10 cells. Furthermore, kuraridin considerably reduced melanogenesis in a 3D human skin model. To elucidate the anti-melanogenic mechanism of kuraridin, target genes (KIT, MAP2K1, and PRKCA) and pathways (c-KIT and ETB-R pathways) were identified using network pharmacology. KIT and MAP2K1 are simultaneously involved in the c-KIT cascade and are considered the most important in melanogenesis. PRKCA acts directly on MITF and its downstream enzymes through another pathway. Docking simulation showed strong interactions between kuraridin and c-KIT, ERK1/2, and PKC encoded by target genes. Overall, the present study showed kuraridin to be a novel natural anti-melanogenic agent in hyperpigmentation disorders.

Endogenous hydrogen peroxide positively regulates secretion of a gut-derived peptide in neuroendocrine potentiation of the oxidative stress response in C. elegans.

The gut-brain axis mediates bidirectional signaling between the intestine and the nervous system and is critical for organism-wide homeostasis. Here we report the identification of a peptidergic endocrine circuit in which bidirectional signaling between neurons and the intestine potentiates the activation of the antioxidant response in C. elegans in the intestine. We identify a FMRF-amide-like peptide, FLP-2, whose release from the intestine is necessary and sufficient to activate the intestinal oxidative stress response by promoting the release of the antioxidant FLP-1 neuropeptide from neurons. FLP-2 secretion from the intestine is positively regulated by endogenous hydrogen peroxide (H2O2) produced in the mitochondrial matrix by sod-3/superoxide dismutase, and is negatively regulated by prdx-2/peroxiredoxin, which depletes H2O2 in both the mitochondria and cytosol. H2O2 promotes FLP-2 secretion through the DAG and calciumdependent protein kinase C family member pkc-2 and by the SNAP25 family member aex-4 in the intestine. Together, our data demonstrate a role for intestinal H2O2 in promoting inter-tissue antioxidant signaling through regulated neuropeptide-like protein exocytosis in a gut-brain axis to activate the oxidative stress response.

Blocking α1 Adrenergic Receptor as a Novel Target for Treating Alzheimer's Disease.

While amyloidopathy and tauopathy have been recognized as hallmarks in Alzheimer's disease (AD) brain, recently, increasing lines of evidence have supported the pathological roles of cerebrovascular changes in the pathogenesis and progression of AD. Restoring or ameliorating the impaired cerebrovascular function during the early phase of the disease may yield benefits against the cognitive decline in AD. In the present study, we evaluated the potential therapeutic effects of nicergoline [NG, a well-known α1 adrenergic receptor (ADR) blocker and vasodilator] against AD through ameliorating vascular abnormalities. Our in vitro data revealed that NG could reverse ß-amyloid1-42 (Aß1-42)-induced PKC/ERK1/2 activation, the downstream pathway of α1-ADR activation, in α1-ADR-overexpressed N2a cells. NG also blocked Aß1-42- or phenylephrine-induced constrictions in isolated rat arteries. All these in vitro data may suggest ADR-dependent impacts of Aß on vascular function and the reversal effect of NG. In addition, the ameliorating impacts of NG treatment on cerebral vasoconstriction, vasoremodeling, and cognitive decline were investigated in vivo in a PSAPP transgenic AD mouse model. Consistent with in vitro findings, the chronic treatment of NG significantly ameliorated the cerebrovascular dysfunctions and Aß plaque depositions in the brain. Moreover, an improved cognitive performance was also observed. Taken together, our findings supported the beneficial effects of NG on AD through adrenergic-related mechanisms and highlighted the therapeutic potential of α1-adrenergic vasomodulators against AD pathologies.

Deciphering the role of PLCD3 in lung cancer: A gateway to glycolytic reprogramming via PKC-Rap1 activation.

PLCD3 belongs to the phospholipase C delta group and is involved in numerous biological functions, including cell growth, programmed cell death, and specialization. However, the role of PLCD3 in lung cancer still needs further investigation. This research aimed to investigate if PLCD3 influences glycolytic reprogramming and lung cancer development through the PKC-dependent Rap1 signaling pathway. This study found that PLCD3 was increased in lung cancer tissues. PLCD3 promotes the proliferation and invasion of lung cancer cells by activating the PKC-dependent Rap1 pathway. The detailed process involves PLCD3 triggering PKC, which subsequently stimulates the Rap1 pathway, leading to glycolytic reprogramming that supplies adequate energy and metabolic substrates necessary for the growth and spread of lung cancer cells. Moreover, PLCD3 can also promote the metastasis and invasion of lung cancer cells by activating the Rap1 pathway. This study reveals the mechanism of PLCD3 in lung cancer and provides new ideas for the treatment of lung cancer. Inhibiting PLCD3, PKC, and the Rap1 pathway may be an effective strategy for treating lung cancer.

The Predictive Value of PKC and ET-1 Levels in Cerebrospinal Fluid for Vasospasm and Prognosis in Patients with Aneurysmal Subarachnoid Hemorrhage.

Objective: To analyze the predictive value of protein kinase C (PKC) and endothelin-1 (ET-1) in cerebrospinal fluid for vasospasm and prognosis in patients with aneurysmal subarachnoid hemorrhage (ASH). Methods: One hundred and forty-eight ASH patients hospitalized in our hospital during February 2019 to February 2022 were optioned as observation subjects. These subjects were graded into good prognosis group (mRS score 0-2, n = 102) and poor prognosis group (mRS score 3-6, n = 46) according to the Rankin Revised Scale Score (mRS) after 6 months of follow-up. Cerebrospinal fluid was collected from patients to detect the content of ET-1 and PKC. The prognostic factors were analyzed using multifactorial logistic regression. The predictive value was assessed using receiver operating characteristic (ROC) curve. Results: The patients with poor prognosis had a higher age level and a higher proportion of ≥2 aneurysms, aneurysm diameter ≥6 mm, cerebral vasospasm, and Hunt-Hess grade ≥III than those with good prognosis (P < 0.05). The patients with poor prognosis had higher content of PKC and ET-1 than those with good prognosis (P < 0.05). Age, aneurysm diameter ≥6 mm, cerebral vasospasm, Hunt-Hess classification ≥grade III, PKC and ET-1 were all risk factors related to the prognosis of ASH (P < 0.05). The area under the curve (AUC) of PKC and ET-1 for diagnosing poor prognosis of ASH was 0.803 and 0.720, respectively. The AUC of the combined detection was 0.873 (P < 0.05). Patients with cerebrovascular spasm had higher content of PKC and ET-1 than those without (P < 0.05). The AUC of PKC and ET-1 for diagnosing cerebral vasospasm in ASH was 0.891 and 0.816, respectively, which was 0.932 for combined detection (P < 0.05). Conclusion: The combination of PKC and ET-1 in cerebrospinal fluid had certain value in predicting the poor prognosis of patients with ASH.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

PKC-mediated phosphorylation governs the stability and function of CELF1 as a driver of EMT in breast epithelial cells.

Epithelial to mesenchymal transition (EMT) is believed to be a principal factor contributing to cancer metastasis. The post-transcriptional and post-translational mechanisms underlying EMT are comparatively underexplored. We previously demonstrated that the CELF1 RNA binding protein is necessary and sufficient to drive the EMT of breast epithelial cells, and that the relative protein expression of CELF1 in this context was dictated at the post-translational level. Here, we elucidate the mechanism of this regulation. Mass spectrometric analysis of CELF1 isolated from mesenchymal MCF-10A cells identified multiple sites of serine and threonine phosphorylation on the protein, correlating with the increased stability of this protein in this cellular state. Analysis of phosphomimetic and serine/threonine-to-alanine phosphomutant variants of CELF1 revealed that these phosphorylation sites indeed dictate CELF1 stability, ubiquitination state, and function in vitro. Via co-immunoprecipitation and in vitro kinase assays, we identified the Protein Kinase C (PKC) alpha and epsilon isozymes as the kinases responsible for CELF1 phosphorylation in a breast cell line. Genetic epistasis experiments confirmed that these PKCs function upstream of CELF1 in this EMT program, and CELF1 phosphorylation impacts tumor metastasis in a xenograft model. This work is the first to formally establish the mechanisms underlying post-translational control of CELF1 expression and function during EMT of breast epithelial cells. Given the broad dysregulation of CELF1 expression in human breast cancer, our results may ultimately provide knowledge that may be leveraged for novel therapeutic interventions in this context.

An Ingenane-Type Diterpene from Euphorbia kansui Promoted Cell Apoptosis and Macrophage Polarization via the Regulation of PKC Signaling Pathways.

Euphorbia kansui, a toxic Chinese medicine used for more than 2000 years, has the effect of "purging water to promote drinking" and "reducing swelling and dispersing modules". Diterpenes and triterpenes are the main bioactive components of E. kansui. Among them, ingenane-type diterpenes have multiple biological activities as a protein kinase C δ (PKC-δ) activator, which have previously been shown to promote anti-proliferative and pro-apoptotic effects in several human cancer cell lines. However, the activation of PKC subsequently promoted the survival of macrophages. Recently, we found that 13-hydroxyingenol-3-(2,3-dimethylbutanoate)-13-dodecanoate (compound A) from E. kansui showed dual bioactivity, including the inhibition of tumor-cell-line proliferation and regulation of macrophage polarization. This study identifies the possible mechanism of compound A in regulating the polarization state of macrophages, by regulating PKC-δ-extracellular signal regulated kinases (ERK) signaling pathways to exert anti-tumor immunity effects in vitro, which might provide a new treatment method from the perspective of immune cell regulation.

Nonalcoholic Fatty Liver Disease and Staging of Hepatic Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is in parallel with the obesity epidemic, and it is the most common cause of liver diseases. The patients with severe insulin-resistant diabetes having high body mass index (BMI), high-grade adipose tissue insulin resistance, and high hepatocellular triacylglycerols (triglycerides; TAG) content develop hepatic fibrosis within a 5-year follow-up. Insulin resistance with the deficiency of insulin receptor substrate-2 (IRS-2)-associated phosphatidylinositol 3-kinase (PI3K) activity causes an increase in intracellular fatty acid-derived metabolites such as diacylglycerol (DAG), fatty acyl CoA, or ceramides. Lipotoxicity-related mechanism of NAFLD could be explained still best by the "double-hit" hypothesis. Insulin resistance is the major mechanism in the development and progression of NAFLD/nonalcoholic steatohepatitis (NASH). Metabolic oxidative stress, autophagy, and inflammation induce NASH progression. In the "first hit" the hepatic concentrations of diacylglycerol increase with an increase in saturated liver fat content in human NAFLD. Activities of mitochondrial respiratory chain complexes are decreased in the liver tissue of patients with NASH. Hepatocyte lipoapoptosis is a critical feature of NASH. In the "second hit," reduced glutathione levels due to oxidative stress lead to the overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling that induces cell death in the steatotic liver. Accumulation of toxic levels of reactive oxygen species (ROS) is caused at least by two ineffectual cyclical pathways. First is the endoplasmic reticulum (ER) oxidoreductin (Ero1)-protein disulfide isomerase oxidation cycle through the downstream of the inner membrane mitochondrial oxidative metabolism and the second is the Kelch like-ECH-associated protein 1 (Keap1)-nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. In clinical practice, on ultrasonographic examination, the elevation of transaminases, γ-glutamyltransferase, and the aspartate transaminase to platelet ratio index indicates NAFLD. Fibrosis-4 index, NAFLD fibrosis score, and cytokeratin18 are used for grading steatosis, staging fibrosis, and discriminating the NASH from simple steatosis, respectively. In addition to ultrasonography, "controlled attenuation parameter," "magnetic resonance imaging proton-density fat fraction," "ultrasound-based elastography," "magnetic resonance elastography," "acoustic radiation force impulse elastography imaging," "two-dimensional shear-wave elastography with supersonic imagine," and "vibration-controlled transient elastography" are recommended as combined tests with serum markers in the clinical evaluation of NAFLD. However, to confirm the diagnosis of NAFLD, a liver biopsy is the gold standard. Insulin resistance-associated hyperinsulinemia directly accelerates fibrogenesis during NAFLD development. Although hepatocyte lipoapoptosis is a key driving force of fibrosis progression, hepatic stellate cells and extracellular matrix cells are major fibrogenic effectors. Thereby, these are pharmacological targets of therapies in developing hepatic fibrosis. Nonpharmacological management of NAFLD mainly consists of two alternatives: lifestyle modification and metabolic surgery. Many pharmacological agents that are thought to be effective in the treatment of NAFLD have been tried, but due to lack of ability to attenuate NAFLD, or adverse effects during the phase trials, the vast majority could not be licensed.

Endothelial Dysfunction in Obesity and Therapeutic Targets.

Parallel to the increasing prevalence of obesity in the world, the mortality from cardiovascular disease has also increased. Low-grade chronic inflammation in obesity disrupts vascular homeostasis, and the dysregulation of adipocyte-derived endocrine and paracrine effects contributes to endothelial dysfunction. Besides the adipose tissue inflammation, decreased nitric oxide (NO)-bioavailability, insulin resistance (IR), and oxidized low-density lipoproteins (oxLDLs) are the main factors contributing to endothelial dysfunction in obesity and the development of cardiorenal metabolic syndrome. While normal healthy perivascular adipose tissue (PVAT) ensures the dilation of blood vessels, obesity-associated PVAT leads to a change in the profile of the released adipo-cytokines, resulting in a decreased vasorelaxing effect. Higher stiffness parameter ß, increased oxidative stress, upregulation of pro-inflammatory cytokines, and nicotinamide adenine dinucleotide phosphate (NADP) oxidase in PVAT turn the macrophages into pro-atherogenic phenotypes by oxLDL-induced adipocyte-derived exosome-macrophage crosstalk and contribute to the endothelial dysfunction. In clinical practice, carotid ultrasound, higher leptin levels correlate with irisin over-secretion by human visceral and subcutaneous adipose tissues, and remnant cholesterol (RC) levels predict atherosclerotic disease in obesity. As a novel therapeutic strategy for cardiovascular protection, liraglutide improves vascular dysfunction by modulating a cyclic adenosine monophosphate (cAMP)-independent protein kinase A (PKA)-AMP-activated protein kinase (AMPK) pathway in PVAT in obese individuals. Because the renin-angiotensin-aldosterone system (RAAS) activity, hyperinsulinemia, and the resultant IR play key roles in the progression of cardiovascular disease in obesity, RAAS-targeted therapies contribute to improving endothelial dysfunction. By contrast, arginase reciprocally inhibits NO formation and promotes oxidative stress. Thus, targeting arginase activity as a key mediator in endothelial dysfunction has therapeutic potential in obesity-related vascular comorbidities. Obesity-related endothelial dysfunction plays a pivotal role in the progression of type 2 diabetes (T2D). The peroxisome proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (thiazolidinedione), is a popular drug for treating diabetes; however, it leads to increased cardiovascular risk. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin (EMPA) significantly improves endothelial dysfunction and mortality occurring through redox-dependent mechanisms. Although endothelial dysfunction and oxidative stress are alleviated by either metformin or EMPA, currently used drugs to treat obesity-related diabetes neither possess the same anti-inflammatory potential nor simultaneously target endothelial cell dysfunction and obesity equally. While therapeutic interventions with glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide or bariatric surgery reverse regenerative cell exhaustion, support vascular repair mechanisms, and improve cardiometabolic risk in individuals with T2D and obesity, the GLP-1 analog exendin-4 attenuates endothelial endoplasmic reticulum stress.

Dendritic cell-specific deletion of PKCδ in offspring of allergic mothers prevents the predisposition for development of allergic lung inflammation in offspring.

In humans and in mice, maternal allergy predisposes offspring to development of allergy. In murine models, increased levels of maternal ß-glucosylceramides are both necessary and sufficient for the development of allergic predisposition in offspring. Furthermore, increased numbers of CD11b+ dendritic cell subsets in the offspring of allergic mothers are associated with allergic predisposition. In vitro, ß-glucosylceramides increase CD11b+ dendritic cell subset numbers through increased PKCδ signaling but it is not known if enhanced PKCδ signaling in dendritic cells is required in vivo. We demonstrate that dendritic cell-specific deletion of PKCδ prevents the ß-glucosylceramide-induced increase in CD11b+ dendritic cell subset numbers both in vitro as well as in vivo in the fetal liver of offspring of mothers injected with ß-glucosylceramides. Furthermore, dendritic cell-specific deletion of PKCδ in offspring prevents the maternal allergy-induced increase in CD11b+ dendritic cell subsets and decreases allergen-induced IL-5 and eosinophilia in lungs of offspring. However, loss of PKCδ in dendritic cells did not prevent development of allergen-specific IgE. Our study provides mechanistic insight into the function of PKCδ in the origins of allergic disease beginning in utero as well as the development of postnatal allergic lung inflammation.

Vitamin D3 Attenuates Neuropathic Pain via Suppression of Mitochondria-Associated Ferroptosis by Inhibiting PKCα/NOX4 Signaling Pathway.

AIMS: Neuropathic pain remains a significant unmet medical challenge due to its elusive mechanisms. Recent clinical observations suggest that vitamin D (VitD) holds promise in pain relief, yet its precise mechanism of action is still unclear. This study explores the therapeutical role and potential mechanism of VitD3 in spared nerve injury (SNI)-induced neuropathic pain rat model. METHODS: The analgesic effects and underlying mechanisms of VitD3 were evaluated in SNI and naïve rat models. Mechanical allodynia was assessed using the Von Frey test. Western blotting, immunofluorescence, biochemical assay, and transmission electron microscope (TEM) were employed to investigate the molecular and cellular effects of VitD3. RESULTS: Ferroptosis was observed in the spinal cord following SNI. Intrathecal administration of VitD3, the active form of VitD, activated the vitamin D receptor (VDR), suppressed ferroptosis, and alleviated mechanical nociceptive behaviors. VitD3 treatment preserved spinal GABAergic interneurons, and its neuroprotective effects were eliminated by the ferroptosis inducer RSL3. Additionally, VitD3 mitigated aberrant mitochondrial morphology and oxidative metabolism in the spinal cord. Mechanistically, VitD3 inhibited SNI-induced activation of spinal PKCα/NOX4 signaling. Inhibition of PKCα/NOX4 signaling alleviated mechanical pain hypersensitivity, accompanied by reduced ferroptosis and mitochondrial dysfunction in SNI rats. Conversely, activation of PKCα/NOX4 signaling in naïve rats induced hyperalgesia, ferroptosis, loss of GABAergic interneurons, and mitochondrial dysfunction in the spinal cord, all of which were reversed by VitD3 treatment. CONCLUSIONS: Our findings provide evidence that VitD3 attenuates neuropathic pain by preserving spinal GABAergic interneurons through the suppression of mitochondria-associated ferroptosis mediated by PKCα/NOX4 signaling, probably via VDR activation. VitD, alone or in combination with existing analgesics, presents an innovative therapeutic avenue for neuropathic pain.

Different Numbers of Conjunctive Stimuli Induce LTP or LTD in Mouse Cerebellar Purkinje Cell.

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses plays an important role in cerebellum-related motor coordination and learning. LTD is induced by the conjunction of PF stimulation and climbing fiber (CF) stimulation or somatic PC depolarization, while long-term potentiation (LTP) is induced by PF stimulation alone. Therefore, it is considered that different types of stimulation induce different types of synaptic plasticity. However, we found that a small number of conjunctive stimulations (PF + somatic depolarization of PC) induced LTP, but did not induce LTD of a small size. This LTP was not associated with changes in paired-pulse ratio, suggesting postsynaptic origin. Additionally this LTP was dependent on nitric oxide. This LTP was also induced by a smaller number of physiological conjunctive PF and CF stimuli. These results suggested that a larger number or longer period of conjunctive stimulation is required to induce LTD by overcoming LTP. Ca2+ transients at the PC dendritic region was measured by calcium imaging during LTD-inducing conjunctive stimulation. Peak amplitude of Ca2+ transients increased gradually during repetitive conjunctive stimulation. Instantaneous peak amplitude was not different between the early phase and late phase, but the average amplitude was larger in the later phase than in the early phase. These results show that LTD overcomes LTP, and increased Ca2+ integration or a number of stimulations is required for LTD induction.

Angong Niuhuang Wan ameliorates LPS-induced cerebrovascular edema by inhibiting blood‒brain barrier leakage and promoting the membrane expression of AQP4.

Introduction: Angong Niuhuang Wan (AGNHW), developed during the Qing dynasty (18th century) for the treatment of consciousness disturbances caused by severe infections, has been used to treat brain edema caused by ischemia‒reperfusion. However, it remains unclear whether AGNHW can ameliorate vascular-origin brain edema caused by lipopolysaccharides (LPS). This study explored the ameliorative effects of AGNHW on LPS-induced cerebrovascular edema in mice, as well as the potential underlying mechanisms. Methods: A cerebrovascular edema model was established in male C57BL/6N mice by two intraperitoneal injections of LPS (15 mg/kg), at 0 and 24 h. AGNHW was administered by gavage at doses of 0.2275 g/kg, 0.455 g/kg, and 0.91 g/kg, 2 h after LPS administration. In control mice, normal saline (NS) or AGNHW (0.455 g/kg) was administered by gavage 2 h after intraperitoneal injection of NS. The survival rate, cerebral water content, cerebral venous FITC-dextran leakage, Evans blue extravasation, and expression of vascular endothelial cadherin (VE-cadherin), zonula occludens-1 (ZO-1), claudin-5, phosphorylated caveolin-1 (CAV-1), and cytomembrane and cytoplasmic aquaporin 1 (AQP1) and aquaporin 4 (AQP4) were evaluated. The cerebral tissue phosphoproteome, blood levels of AGNHW metabolites, and the relationships between these blood metabolites and differentially phosphorylated proteins were analyzed. Results: AGNHW inhibited the LPS-induced decrease in survival rate, increase in cerebral water content, decrease in VE-Cadherin expression and increase in phosphorylated CAV-1 (P-CAV-1). AGNHW treatment increased the expression of AQP4 on astrocyte membrane after LPS injection. AGNHW also inhibited the LPS-induced increases in the phosphorylation of 21 proteins, including protein kinase C-α (PKC-α) and mitogen-activated protein kinase 1 (MAPK1), in the cerebral tissue. Eleven AGNHW metabolites were detected in the blood. These metabolites might exert therapeutic effects by regulating PKC-α and MAPK1. Conclusion: AGNHW can ameliorate cerebrovascular edema caused by LPS. This effect is associated with the inhibition of VE-Cadherin reduction and CAV-1 phosphorylation, as well as the upregulation of AQP4 expression on the astrocyte membrane, following LPS injection.

PKCα Induced the Generation of Extracellular Vesicles in Activated Platelets to Promote Breast Cancer Metastasis.

Platelet extracellular vesicles (PEVs) play an important role in tumor development. However, the mechanisms underlying their biogenesis have not been fully elucidated. Protein kinase Cα (PKCα) is an important regulator of platelet activation, but the effect of PKCα on EV generation is unclear. We used small-particle flow cytometry and found that the number of PEVs was increased in patients with breast cancer compared to those with benign breast disease. This was accompanied by increased levels of activated PKCα in breast cancer platelets. Treating platelets with the PKCα agonist phorbol 12-myristate 13-acetate (PMA) increased the phosphorylation PKCα and induced PEV production, while the PKCα inhibitor GÖ6976 showed the opposite effects. Notably, incubating platelets from patients with benign tumors with the culture supernatant of MDA-MB-231 cells induced PKCα phosphorylation in the platelets. Mass spectrometry and coimmunoprecipitation assays showed that Dynamin 2 (DNM2), a member of the guanosine-triphosphate-binding protein family, might cooperate with activated PKCα to regulate PEV production by breast cancer platelets. Similar results were observed in a mouse model of lung metastasis. In addition, PEVs were engulfed by breast cancer cells and promoted cancer cell migration and invasion via miR-1297 delivery. These findings suggested that PKCα cooperates with DNM2 to induce PEV generation, and PEV release might triggered by factors in the breast cancer environment.

The Anti-Metastatic Action of Oxyresveratrol via Suppression of Phosphoryl-ERK/-PKCα-Mediated Sp1/MMP1 Signaling in Human Renal Carcinoma Cells.

Oxyresveratrol (OxyR) exerts biological and pharmacological effects in a variety of tumor cells, including antioxidant action, antitumor activity, and proapoptotic effects. However, the regulation of targeted signaling pathways by OxyR and the mechanism underlying these effects in human renal cell carcinoma (RCC) have been less studied. We observed that OxyR at noncytotoxic doses did not affect the growth of human RCC cells or normal kidney HK2 cells. OxyR inhibited ACHN and Caki-1 cell migration and invasion through targeting matrix metalloproteinase 1 (MMP1) expression. Analysis of clinical databases showed that high MMP1 expression is associated with lower overall survival (OS) in these cancers (p < 0.01). OxyR significantly inhibited the mRNA and protein expression of Sp1. Furthermore, luciferase assay results showed that OxyR inhibited Sp1 transcriptional activity. Additionally, OxyR preferentially suppressed the activation of ERK and PKCα. Treatment with U0126 (MEK inhibitor) or G06976 (PKCα inhibitor) clearly decreased Sp1 and MMP1 expression and inhibited RCC cell migration and invasion. In conclusion, OxyR may be a potential antitumor therapy for the inhibition of migration and invasion by controlling p-ERK/Sp1 and p-PKCα/Sp1-mediated MMP1 expression in RCC.

Targeting HTR2B suppresses non-functioning pituitary adenoma growth and sensitizes cabergoline treatment via inhibiting Gαq/PLC/PKCγ/STAT3 axis.

BACKGROUND: Managing non-functioning pituitary adenomas (NFPAs) is difficult due to limited drug treatments. Cabergoline's (CAB) effectiveness for NFPAs is debated. This study explores the role of HTR2B in NFPAs and its therapeutic potential. METHODS: We conducted screening of bulk RNA-sequencing data to analyze HTR2B expression levels in NFPA samples. In vitro and in vivo experiments were performed to evaluate the effects of HTR2B modulation on tumor growth and cell cycle regulation. Mechanistic insights into the HTR2B-mediated signaling pathway were elucidated using pharmacological inhibitors and molecular interaction assays. RESULTS: Elevated HTR2B expression was detected in NFPA samples, which was associated with increased tumor survival. Inhibition of HTR2B activity resulted in the suppression of tumor growth through modulation of the G2M cell cycle. The inhibition of HTR2B with PRX-08066 was found to block STAT3 phosphorylation and nuclear translocation by interfering with the Gαq/PLC/PKC pathway. A direct interaction between PKC-γ and STAT3 was critical for STAT3 activation. CAB was shown to activate pSTAT3 via HTR2B, reducing its therapeutic potential. However, the combination of an HTR2B antagonist with CAB significantly inhibited tumor cell proliferation in HTR2B-expressing pituitary tumor cell lines, a xenografted pituitary tumor model, and patient-derived samples. Analysis of patient-derived data indicated that a distinct molecular pattern characterized by upregulated HTR2B/PKC-γ and downregulated BTG2/GADD45A may benefit from combination treatment with CAB and PRX-08066. CONCLUSIONS: HTR2B is a potential therapeutic target for NFPAs, and its inhibition could improve CAB efficacy. A dual therapy approach may be beneficial for NFPA patients with high HTR2B expression.

ICA1 affects APP processing through the PICK1-PKCα signaling pathway.

AIMS: Islet cell autoantigen 1 (ICA1) is involved in autoimmune diseases and may affect synaptic plasticity as a neurotransmitter. Databases related to Alzheimer's disease (AD) have shown decreased ICA1 expression in patients with AD. However, the role of ICA1 in AD remains unclear. Here, we report that ICA1 expression is decreased in the brains of patients with AD and an AD mouse model. RESULTS: The ICA1 increased the expression of amyloid precursor protein (APP), disintegrin and metalloprotease 10 (ADAM10), and disintegrin and metalloprotease 17 (ADAM17), but did not affect protein half-life or mRNA levels. Transcriptome sequencing analysis showed that ICA1 regulates the G protein-coupled receptor signaling pathway. The overexpression of ICA1 increased PKCα protein levels and phosphorylation. CONCLUSION: Our results demonstrated that ICA1 shifts APP processing to non-amyloid pathways by regulating the PICK1-PKCα signaling pathway. Thus, this study suggests that ICA1 is a novel target for the treatment of AD.