PKCδ Expression Research Articles

Regulation of pro-apoptotic and anti-apoptotic factors in obesity-related esophageal adenocarcinoma

BackgroundObesity is a risk factor for esophageal adenocarcinoma (EAC). It was reported that obesity -associated inflammation correlates with insulin resistance and increased risk of EAC. The objective of the study is to investigate the role of obesity associated inflammatory mediators in the development of EAC.MethodsWe included 23 obese and nonobese patients with EAC or with or without Barrett’s esophagus (BE) after IRB approval. We collected 23 normal, 10 BE, and 19 EAC tissue samples from endoscopy or esophagectomy. The samples were analyzed for the expression levels of pro-apoptotic and anti-apoptotic factors, PKC-δ, cIAP2, FLIP, IGF-1, Akt, NF-kB and Ki67 by immunofluorescence and RT-PCR. We compared the expression levels between normal, BE, and EAC tissue using Students’ t-test between two groups.ResultsOur results showed decreased gene and protein expression of pro-apoptotic factors (bad, bak and bax) and increased expression of anti-apoptotic factors (bcl-2, Bcl-xL) in BE and EAC compared to normal tissues. There was increased gene and protein expression of PKC-δ, cIAP2, FLIP, NF-kB, IGF-1, Akt, and Ki67 in BE and EAC samples compared to normal esophagus. Further, an increased folds changes in mRNA expression of proapoptotic factors, antiapoptotic factors, PKC-δ, IGF-1, Akt, and Ki-67 was associated with obesity.ConclusionPatients with EAC had increased expression of cIAP2 and FLIP, and PKC-δ which is associated with inhibition of apoptosis and possible progression of esophageal adenocarcinoma.

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/β-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and β-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and β-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on β-catenin in the canonical Wnt pathway was limited. β-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Protein Kinase C-Delta Mediates Cell Cycle Reentry and Apoptosis Induced by Amyloid-Beta Peptide in Post-Mitotic Cortical Neurons.

Amyloid-beta peptide (Aβ) is a neurotoxic constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. The detailed mechanisms by which protein kinase C-delta (PKCδ) contributes to Aβ toxicity is not yet entirely understood. Using fully differentiated primary rat cortical neurons, we found that inhibition of Aβ25-35-induced PKCδ increased cell viability with restoration of neuronal morphology. Using cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) as the respective markers for the G1-, S-, and G2/M-phases, PKCδ inhibition mitigated cell cycle reentry (CCR) and subsequent caspase-3 cleavage induced by both Aβ25-35 and Aβ1-42 in the post-mitotic cortical neurons. Upstream of PKCδ, signal transducers and activators of transcription (STAT)-3 mediated PKCδ induction, CCR, and caspase-3 cleavage upon Aβ exposure. Downstream of PKCδ, aberrant neuronal CCR was triggered by overactivating cyclin-dependent kinase-5 (CDK5) via calpain2-dependent p35 cleavage into p25. Finally, PKCδ and CDK5 also contributed to Aβ25-35 induction of p53-upregulated modulator of apoptosis (PUMA) in cortical neurons. Together, we demonstrated that, in the post-mitotic neurons exposed to Aβs, STAT3-dependent PKCδ expression triggers calpain2-mediated p35 cleavage into p25 to overactivate CDK5, thus leading to aberrant CCR, PUMA induction, caspase-3 cleavage, and ultimately apoptosis.

PKCδ serves as a potential biomarker and therapeutic target for microglia-mediated neuroinflammation in Alzheimer's disease.

To investigate the role of a novel type of protein kinase C delta (PKCδ) in the neuroinflammation of Alzheimer's disease (AD). We analyzed PKCδ and inflammatory cytokines levels in cerebrospinal fluid (CSF) of AD and normal controls, as well as their correlations. The cellular expression pattern of PKCδ and the effects of PKCδ modulation on microglia-mediated neuroinflammation were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, RNA sequencing (RNA-seq), and immunofluorescence staining. PKCδ levels were increased dramatically in the CSF of AD patients and positively correlated with cytokines. PKCδ is expressed mainly in microglia in the brain. Amyloid beta (Aβ) stimulation increased PKCδ expression and secretion, which led to upregulation of the nuclear factor kappa B (NF-κB) pathway and overproduction of proinflammatory cytokines. Downregulation or inhibition of PKCδ attenuated Aβ-induced microglial responses and improved cognitive function in an AD mouse model. Our study identifies PKCδ as a potential biomarker and therapeutic target for microglia-mediated neuroinflammation in AD. Protein kinase C delta (PKCδ) levels increase in cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD), and positively correlate with elevated inflammatory cytokines in human subjects. PKCδ is expressed mainly in microglia in vivo, whereas amyloid beta (Aβ) stimulation increases PKCδ expression and secretion, causing upregulation of the nuclear factor kappa B (NF-κB) pathway and production of inflammatory cytokines. Downregulation or inhibition of PKCδ attenuates Aβ-enhanced NF-κB signaling and cytokine production in microglia and improves cognitive function in AD mice. PKCδ serves as a potential biomarker and therapeutic target for microglia-mediated neuroinflammation in AD.

Upregulation of rate-limiting enzymes in cholesterol metabolism by PKCδ mediates endothelial apoptosis in diabetic wound healing

Diabetic foot ulcer (DFU) is a prevalent complication of diabetes that poses significant challenges in terms of treatment and management. It is characterized by heightened endothelial apoptosis and impaired angiogenesis. In this study, we aimed to investigate the role of protein kinase Cδ (PKCδ) in regulating endothelial apoptosis in diabetic wounds by promoting cholesterol biosynthesis. The expression of PKCδ was increased in human umbilical vascular endothelial cells (HUVECs) cultivated in high glucose medium and skin tissue isolated from diabetic mice. High glucose-induced HUVECs apoptosis was reduced by PKCδ inhibition with siRNA or rottlerin. RNA-seq identified two enzymes, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), as the downstream of PKCδ. PKCδ knockdown or inhibition suppressed the expression of HMGCS1 and HMGCR and lowered free cholesterol (FC) levels. Cholesterol restored high glucose-induced apoptosis in siRNA- or rottlerin-treated HUVECs. In vivo use of rosuvastatin calcium, an inhibitor of HMGCR, downregulated free cholesterol levels and accelerated the wound healing process. In conclusion, PKCδ expression in endothelial cells was activated by high glucose, which subsequently upregulates the expression of two enzymes catalyzing cholesterol biosynthesis, HMGCS1 and HMGCR. Enhanced cholesterol biosynthesis raises free cholesterol levels, promotes endothelial apoptosis, and finally delays wound healing.

Cardioprotective effect of cemtirestat treatment in male Zucker diabetic fatty rats: a pilot study

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Slovak Research and Development under the contract no. 21-0410 (APVV) and 20-0411 (APVV) and VEGA grant no. 2/0006/23 Introduction Type 2 Diabetes Mellitus (T2DM), the most common metabolic disorder in human population is caused by defective insulin secretion and reduced tissue responsiveness to insulin. T2DM is characterized by hyperglycaemia accompanied by oxidative stress and inflammation that results in organopathy. The Zucker diabetic fatty rats (ZDF) are established animal model for research of T2DM associated with obesity, and for efficacy of novel treatments. Myocardial electrical coupling protein, connexin43 (Cx43) is one of the key factors affecting heart function and arrhythmogenesis. We have previously shown upregulation of Cx43 in type 1 DM rats that was associated with reduced susceptibility to malignant arrhythmias. However, it is not known whether T2DM affects Cx43 expression and/or function in the heart as well as in brown adipose tissue (BAT). The purpose of our study was to examine expression of Cx43 in heart and BAT as well as the effect of treatment with highly selective and efficient aldose reductase inhibitor enriched with antioxidant activity, Cemtirestat. Methods: Forty 4-month-old male ZDF rats (fa/+ and fa/fa) were divided into 4 groups: 1) untreated control lean rats (fa/+), 2) control lean rats treated with Cemtirestat, 3) untreated diabetic fatty rats (fa/fa), 4) diabetic fatty rats treated with Cemtirestat (2,55 mg/kg/day). After 6 months of treatment, biometric parameters were registered and left ventricle (LV) + BAT were used for Western blot analysis of Cx43, PKCɛ (phosphorylates Cx43) and PKCδ (implicated in pro­apoptotic signaling). BAT and LV tissue sections were also used for immunolabeling of Cx43 to examine its topology. Results: Increase of body weight, heart weight, LV weight, adiposity, plasma triglycerides, cholesterol and blood glucose were registered in ZDF fa/fa rats. T2DM resulted in altered localisation of Cx43 that was not affected by treatment with Cemtirestat. Expression of Cx43 was significantly decreased in LV of fa/fa rats and restored with Cemtirestat. Moreover, BAT exhibited significantly lower expression of total Cx43 in fa/fa rats, that was moderately increased by Cemtirestat. The expression of PKCɛ was significantly decreased in both, LV and BAT of fa/fa rats with mild elevation of PKCɛ in LV of treated fa/fa rats. Cemtirestat slightly normalized increased expression of PKCδ in LV of fa/fa rats, but had no effect on lower PKCδ levels in BAT of fa/fa rats. Echocardiographic examination did not show significant difference between treated and untreated groups of rats and showed mild differences between diabetic and control rats. In conclusions, findings point out that T2DM downregulates Cx43 in the heart and BAT, however further studies are necessary for understanding the mechanisms implicated in every step in the development of T2DM cardiopathy and arrhythmogenesis as well as BAT alterations. Further research is required to determine factors involved in cardioprotective effects of Cemtirestat.

Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes.

Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.

Abstract 14353: Deficiency of Protein Kinase Cδ Revamps Atherosclerotic Plaque Complexity by Halting Inflammasome Activation and Intensifying Mitochondrial Function

Introduction: Plaque complexities substantially contribute to acute coronary syndrome. Inflammation and cell death in macrophages play a vital role in the formation of vulnerable plaques. Previous study had shown that protein kinase Cδ (PKCδ) is involved in the initiation and progression of atherosclerosis. However, the role of PKCδ on plaque stability remains unclear. Hypothesis: PKCδ regulates macrophage inflammation and cell death, leading to the formation of vulnerable plaques. Methods: The PKC family gene expressions in human atheroma plaques and nearby macroscopically intact tissues were analyzed from the gene expression omnibus data sets GSE43292. Two main mice models, including Ldlr -/- / PKCδ -/- mice and Ldlr -/- mice lethally irradiated and bone marrow transplanted (BMT) with either PKCδ -/- or wild type (WT), were applied to evaluate the impact of macrophages PKCδ on atherosclerosis. Blood, aorta, and aortic sinus were collected. The effects and underlying molecular mechanisms of PKCδ on inflammasome activation, pyroptosis, and mitochondrial function were explored using bone marrow-derived macrophage (BMDM) from PKCδ -/- and WT mice. Results: The gene expression of PKCδ significantly increases in human atheroma plaques, which is correlated with the expression of NLRP3 inflammasome. The deletion of PKCδ reduces plasma lipid profile and atherosclerosis in Ldlr -/- mice. Moreover, the deletion of PKCδ attenuates necrotic core formation, in both double knockout and BMT models. The RNA-seq analysis revealed that pyroptosis and mitochondrial oxidative phosphorylation (OXPHOS) are the potential mechanisms in PKCδ-regulated atherosclerosis. Additionally, knockout of PKCδ reduces expressions of LPS/IFNγ-induced TNFα, IL-6, and NLRP3 inflammasome in BMDM. Finally, the deficiency of PKCδ increases mitochondrial OXPHOS in BMDM, suggesting a potential role for PKCδ in the regulation of cellular metabolism and inflammation. Conclusions: Our findings indicate a crucial role for PKCδ in the development of complex atherosclerotic plaques, as it promotes inflammation, pyroptosis, and mitochondrial dysfunction. Targeting PKCδ could potentially be a therapeutic strategy for the treatment of atherosclerosis.

Apelin receptor inhibition in ischemia-reperfused mouse hearts protected by endogenous n-3 polyunsaturated fatty acids.

Background: While the protective effects of n-3 polyunsaturated fatty acids (PUFAs) on cardiac ischemia-reperfusion (IR) injury have been previously reported, limited data are available regarding how these fatty acids affect membrane receptors and their downstream signaling following IR injury. We aimed to identify potential receptors activated by n-3 PUFAs in IR hearts to understand the regulatory mechanisms of these receptors. Methods: We used fat-1 mice, which naturally have elevated levels of n-3 PUFAs, and C57BL/6J mice as a control group to create a myocardial IR injury model through Langendorff perfusion. We assessed the impact of endogenous n-3 PUFAs on left ventricular function, myocardial infarct size, myocardial apoptosis, and ATP production. RNA sequencing (RNA-seq) and bioinformatics analysis were conducted to identify molecular targets affected by n-3 PUFAs. Based on these analyses we then treated IR hearts of WT and fat-1 mice with an antagonist (ML221) or an agonist (apelin-13) for the predicted receptor to assess cardiac contractile function and intracellular signaling pathways. An in vitro hypoxia-reoxygenation (HR) model was also used to confirm the effects of n-3 PUFAs on the examined intracellular signaling pathways. Results: Endogenous n-3 PUFAs protected cardiac structure and function in post-IR hearts, and modulated phosphorylation patterns in the PI3K-AKT-mTOR signaling pathways. RNA-seq analysis revealed that n-3 PUFAs affected multiple biological processes as well as levels of the apelin receptor (APLNR). Consistent with a role for the PLNNR, ML221 synchronized the activation of the PI3K-AKT-mTOR signaling axis, suppressed the expression of PKCδ and phosphorylated p38α, upregulated PKCε expression, upregulated or restored the phosphorylation of myofilaments, and prevented myocardial injury and contractile dysfunction in WT IR hearts. By contrast, apelin-13 disrupted the PI3K-AKT-mTOR signaling axis in post-IR fat-1 hearts. The phosphorylation signaling targeted by APLNR inhibition in post-IR fat-1 hearts was also observed after treating HR cells with eicosatetraenoic acid (EPA). Conclusion: Endogenous n-3 PUFAs protect against post-IR injury and preserve cardiac contractile function possibly through APLNR inhibition. This inhibition synchronizes the PI3K-AKT-mTOR axis, suppresses detrimental phosphorylation signaling, and restores or increases myofilament phosphorylation in post-IR hearts. The beneficial effects observed in fat-1 transgenic mouse hearts can be attributed, at least in part, to elevated EPA levels. This study is the first to demonstrate that n-3 PUFAs protect hearts against IR injury through APLNR inhibition.

Panax Quinquefolium Saponins enhances angiogenesis in rats with diabetes and myocardial infarction

Ethnopharmacological relevanceXi Yang Shen (Panax quinquefolium L.) was originally recorded in “Ben Cao Cong Xin” edited by Wu Yiluo during the Qing Dynasty. Panax Quinquefolium Saponins (PQS) is the main component derived from Panax quinquefolium L, and has been wildly used in the treatment of coronary heart disease. Aim of the studyThis study aims to explore the potential role and underlying mechanisms of PQS in promoting angiogenesis in rats with diabetes and myocardial infarction. Materials and methodsEchocardiograms were used to assess cardiac function, while the heart weight to tibia length ratio was calculated to determine cardiac hypertrophy. Hematoxylin and eosin, periodic acid-Schiff and Masson's trichrome staining were used to examine cardiac morphology, myocyte diameter, and myocardial fibrosis. Immunofluorescence staining was employed to evaluate arteriolar density. The transcriptomes were analyzed and bioinformatic analyses were conducted to predict the potential angiogenesis-promoting mechanism of PQS. In addition, RT-PCR and western blotting was utilized to examine the expression of genes and proteins influenced by PQS. ResultsPQS improved blood glucose, ameliorated cardiac function, reduced cardiac hypertrophy, and enhanced myocardial morphology in diabetic rats with myocardial infarction. PQS was also found to decrease myocyte diameter, curtail myocardial fibrosis, and increase arteriolar density. However, the effects of PQS were abolished following the deletion of protein kinase C δ (PKCδ). Molecular docking predicted strong interactions between the major blood components of PQS and PKCδ. Transcriptomic and bioinformatic analyses indicated that PQS may bolster angiogenesis by activating the VEGF/PI3K-Akt/eNOS pathway in rats with diabetes and myocardial infarction. Finally, the study demonstrated that PQS could inhibit the expression of PKCδ and stimulate the activation of the VEGF/PI3K-Akt/eNOS pathway. ConclusionsPQS improves blood glucose, enhances cardiac function, mitigates cardiac damage, and boosts arteriolar density. The angiogenic impact of PQS appears to be, at least partially, due to its modulation of the PKCδ-mediated VEGF/PI3K-Akt/eNOS signaling pathway in rats with diabetes and myocardial infarction.

PKC-δ Promotes IL-1β-Induced Apoptosis of Rat Chondrocytes and Via Activating JNK and P38 MAPK Pathways.

Protein kinase C-delta (PKC-δ) is involved in apoptosis. This study aimed to establish whether PKC-δ can further promote IL-1β-induced chondrocyte apoptosis by mediating the phosphorylation of the JNK and p38 mitogen-activated protein kinase (MAPK) signaling pathways In osteoarthritis (OA). We employed chondrocyte staining to determine the extent of cartilage degeneration. PKC-δ and p38 signal expressions were used in the immunohistochemical (IHC) test and apoptosis was assayed at the TUNEL test in human osteoarthritic and controls. We stimulated rat cartilage cells using IL-1β (10 ng/ml)/rottlerin (10 μM) or lentivirus. To determine the apoptosis rate, we employed flow cytometry. The mRNA of both BCL2-related X (BAX) and cysteine aspartate protease 3 (caspase-3) could be measured via qRT-PCR. Western blot measured the protein levels of BAX, caspase-3, PKC-δ, p-JNK/JNK and p-p38/p38. The positive rate of PKC-δ and the apoptotic rate of chondrocytes in OA were higher than controls. The manifestation of PKC-δ was positively related to the degree of cartilage degeneration, p38 protein expression, and apoptosis rate. IL-1β exposure upregulated PKC-δ expression in chondrocytes in a dose-dependent manner. Decreasing PKC-δ expression and its phosphorylation in OA can inhibit MAPK signaling pathway activation (phosphorylation) by downregulating JNK and p38 protein phosphorylation and expression. This inhibition decreases caspase-3 and BAX levels, consequently lowering the apoptosis rate in chondrocytes. PKC-δ activation by IL-1β in OA promotes chondrocyte apoptosis via activation of the JNK and p38 MAPK signal pathways, thereby promoting the OA progression.

Nalbuphine attenuates morphine-induced scratching by inhibiting PKCβ-dependent microglial activation and p38 phosphorylation in male mice.

Morphine-induced scratching (MIS) is a common adverse effect associated with the use of morphine as analgesia after surgery. However, the treatment of MIS is less than satisfactory due to its unclear mechanism, which needs to be enunciated. We found that intrathecal (i.t.) injections of morphine significantly enhanced scratching behavior in C57BL/6J male mice as well as increased the expressions of protein kinase C β (PKCβ), phosphorylated p38 mitogen-activated protein kinases (MAPK), and ionized calcium-binding adapter molecule 1 (Iba1) within spinal cord dorsal horn. Conversely, using the kappa opioid receptor antagonist nalbuphine significantly attenuated scratching behavior, reduced PKCβ expression and p38 phosphorylation, and decreased spinal dorsal horn microglial activation, while PKCδ and KOR expression elevated. Spinal PKCβ silencing mitigated MIS and microglial activation. Still, knockdown of PKCδ reversed the inhibitory effect of nalbuphine on MIS and microglial activation, indicating that PKCδ is indispensable for the antipruritic effects of nalbuphine. In contrast, PKCβ is crucial for inducing microglial activation in MIS in male mice. Our findings show a distinct itch cascade of morphine, PKCβ/p38MAPK, and microglial activation, but an anti-MIS pathway of nalbuphine, PKCδ/KOR, and neuron activation.

BNST PKCδ neurons are activated by specific aversive conditions to promote anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a critical mediator of stress responses and anxiety-like behaviors. Neurons expressing protein kinase C delta (BNSTPKCδ) are an abundant but understudied subpopulation implicated in inhibiting feeding, but which have conflicting reports about their role in anxiety-like behaviors. We have previously shown that expression of PKCδ is dynamically regulated by stress and that BNSTPKCδ cells are recruited during bouts of active stress coping. Here, we first show that in vivo activation of this population is mildly aversive. This aversion was insensitive to prior restraint stress exposure. Further investigation revealed that unlike other BNST subpopulations, BNSTPKCδ cells do not exhibit increased cfos expression following restraint stress. Ex vivo current clamp recordings also indicate they are resistant to firing. To elucidate their afferent control, we next used rabies tracing with whole-brain imaging and channelrhodopsin-assisted circuit mapping, finding that BNSTPKCδ cells receive abundant input from affective, arousal, and sensory regions including the basolateral amygdala (BLA) paraventricular thalamus (PVT) and central amygdala PKCδ-expressing cells (CeAPKCδ). Given these findings, we used in vivo optogenetics and fiber photometry to further examine BNSTPKCδ cells in the context of stress and anxiety-like behavior. We found that BNSTPKCδ cell activity is associated with increased anxiety-like behavior in the elevated plus maze, increases following footshock, and unlike other BNST subpopulations, does not desensitize to repeated stress exposure. Taken together, we propose a model in which BNSTPKCδ cells may serve as threat detectors, integrating exteroceptive and interoceptive information to inform stress coping behaviors.

Inhibition of protein kinase C delta leads to cellular senescence to induce anti-tumor effects in colorectal cancer.

Protein kinase C delta (PKCδ) is a multifunctional serine-threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, the molecular mechanism of PKCδ in colorectal cancer (CRC) remains unclear. In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild-type (wt-p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt-p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53-independent pathway and that PKCδ-kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence-like phenotypes, including increased senescence-associated β-galactosidase (SA-β-gal)staining, low LaminB1 expression, large nucleus size, and senescence-associated secretory phenotype (SASP) detection. Finally, a kinase inhibitor of PKCδ suppressed senescence-dependent tumorigenicity in a dose-dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis. In addition, a novel therapeutic strategy for wt-p53 CRC is proposed.

Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

PKCδ promotes the invasion and migration of colorectal cancer through c-myc/NDRG1 pathway.

Colorectal cancer (CRC) is the third cause of expected cancer deaths both in men and women in the U.S. and the third most commonly diagnosed cancer in China Targeted therapy has been proven to improve overall survival for unresectable metastatic CRC. But the location of the primary tumor or the presence of various core driver gene mutations that confer resistance may limit the utility of targeted therapy. Therefore, it is of great significance to further elucidate novel mechanisms of invasion and metastasis of CRC and find potential novel therapeutic targets. Protein Kinase C Delta (PKCδ) plays an important role in various diseases, including tumors. In CRC, the function of PKCδ on proliferation and differentiation is mostly studied but various research results were reported. Therefore, the role of PKCδ in CRC needs to be further studied, especially in tumor invasion and metastasis in CRC which few studies have looked into. The expression of PRKCD was analyzed by the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases and Immunohistochemical (IHC). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) enrichment analysis were used to explore the biological functions and pathways related to PRKCD. Lentivirus transfection was used to construct CRC cell lines with overexpression and knock-down of PKCδ or N-myc Downstream Regulated Gene 1 (NDRG1). Cell invasion and migration assay, wound healing assay were used to detect the function of PKCδ and NDRG1 in the invasion and migration of cells. Flow cytometry analysis was used to detect the influence of PKCδ on the CRC cell cycles .Immunofluorescence histochemistry ,Immunoprecipitation Assay and qPCR were used to detect the relationship of PKCδ and NDRG1. Xenograft model was used to verify the role of PKCδ in vivo. PKCδ is overexpressed in CRC and could promote Epithelial-Mesenchymal Transition (EMT) and the invasion and migration of CRC in vitro. We confirmed that PKCδ and the tumor suppressor factor NDRG1 had a co-localization relationship in CRC. PKCδ inhibited NDRG1 transcription and protein expression. Overexpressing NDRG1 could inhibit the function of PKCδ in promoting tumor invasion and migration. PKCδ could regulate c-Myc, one transcription factor of NDRG1, to down-regulate NDRG1. In vivo, overexpressing PKCδ could promote xenograft growth and volume. Thus, our results showed that PKCδ reduced the expression of NDRG1 through c-Myc, promoting the invasion and migration of CRC through promoting EMT. The increased expression of PKCδ in CRC tumor tissue could promote the invasion and migration of tumor cells, and one of the mechanisms may be regulating c-Myc to inhibit the expression of NDRG1 and promote EMT.

RETRACTED: PEITC: A resounding molecule averts metastasis in breast cancer cells in vitro by regulating PKCδ/Aurora A interplay

Background/aimIntricate association and aberrant activation of serine/threonine kinase (STK) family proteins like Polo-like kinase (PLK1) and Aurora kinase (Aurora A abruptly regulate mitotic entry whereas activation of PKCδ), another important member of STK family conversely induces apoptosis which is preceded by cell cycle arrest. These STKs are considered as major determinant of oncogenicity. Therefore, the contributory role of Aurora A/PLK-1 axis in mitotic control and PKCδ in apoptosis control and their reciprocity in cancer research is an emerging area to explore. The present study investigated the intricate involvement of STKs in breast cancer cells (MCF-7 and MDA-MB-231) and their disruption by PEITC. MethodsBoth MCF-7 and MDA-MB-231 cells were checked for clonogenic assay, cell-cycle analysis and the results were compared with normal MCF-10A, Western blotting, TUNEL & DNA-fragmentation assay, wound healing, transwell migration assays in presence and absence of PEITC. ResultsPEITC was found to increase the expression of PKCδ with subsequent nuclear translocation. Nuclear translocation of PKCδ was accompanied by inhibition of nuclear lamin vis a vis phosphorylation of Nrf2 at Ser 40 alongside nuclear accumulation of phospho-Nrf2. Activated PKCδ furthermore exerted its apoptotic effect by negatively regulating Aurora A and consequentially PLK1; indicating activation of PLK1 by Aurora A. Involvement of PEITC induced PKCδ activation and Aurora A inhibition was ascertained by using Rottlerin/Aurora A Inhibitor. Discussion & conclusionNatural isothiocyanates like PEITC efficiently altered the functional abilities of STKs concerning their entangled functional interplay. Such alterations in protein expression by PEITC was chaperoned with inhibition of the aggressiveness of breast cancer cells and ultimately induction of apoptosis.

Relationship between Protein kinase C isoforms, Telomerase and Alpha-fetoprotein through PI3K/AKT/mTOR pathway in Hepatocellular carcinoma

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

Stem Cell Markers CXCR-4 and CD133 Predict Aggressive Phenotype and Their Double Positivity Indicates Poor Prognosis of Oral Squamous Cell Carcinoma.

Simple SummaryOral cancer is one of the most frequent malignancies in the world, with a poor prognosis. The absence of accurate biomarkers for predicting oral cancer progression is the primary cause of treatment failures. Multiple studies have shown that cancer stem cells play a critical role in tumor growth and chemo resistance. We uncovered, for the first time, the importance of combinatorial expression of stem cell related molecules CXCR-4 and CD-133 as possible biomarkers to predict poor prognosis of oral squamous cell carcinoma. The findings will aid in the identification of high-risk cases in order to provide appropriate therapy.The activation of the SDF-1/CXCR-4 pathway is crucial for the invasion and metastasis of oral cancer cells. The CXCR-4 positive cells possess stem cell characteristics and express the cancer stem cell marker, CD133, in tumors of colon and pancreas. Despite several studies, the co-expression of CXCR-4 and CD133 and its significance is still largely unknown in oral cancer. Therefore, we aimed to investigate the impact of CXCR-4 and CD133 double positivity in the prognosis of oral cancer. The significance of PKC-δ, one of the key signaling molecules that regulates CXCR-4, was also analyzed. Immunohistochemistry and double immunofluorescence was used to investigate the co-localization of CXCR-4, PKC-δ and CD133 in the human tissues and cell lines of oral squamous cell carcinoma. The expression of CXCR-4, PKC-δ and CD133 were found to be higher in poorly differentiated and lymph node metastasis-positive cases. Interestingly, CXCR-4 positive cells showed positive staining for PKC-δ and CD133 in oral cancer tissue and cell lines. Moreover, CXCR-4+/CD133+ and CXCR-4+/PKC-δ+ double positive cases have the worst survival. We discovered, for the first time, that patients with expression of both CXCR-4 and CD133 have a lower survival rate, and CXCR-4+/CD133+, as well as CXCR-4+/PKC-δ+ double positivity, can be utilized to predict poor prognosis. CXCR-4, PKC-δ and CD133 might regulate aggressiveness and invasion of oral cancer cells.

Neuroprotective effect of paeoniflorin in the mouse model of Parkinson's disease through α-synuclein/protein kinase C δ subtype signaling pathway.

Paeoniflorin, an active component of Radix Paeoniae Alba, has a neuroprotective effect in Parkinson's animal models. However, its mechanism of action remains to be determined. In this study, we hypothesized that the neuroprotective effect of paeoniflorin occurs through the α-synuclein/protein kinase C δ subtype (PKC-δ) signaling pathway. We tested our hypothesis in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease. We evaluated the effects of paeoniflorin on the expression levels of signal components of the α-synuclein/PKC-δ pathway, cellular apoptosis and motor performance. Our results demonstrated that paeoniflorin restored the motor performance impairment caused by MPTP, inhibited apoptosis, and protected the ultrastructure of neurons. Paeoniflorin treatment also resulted in the dose-dependent upregulation of an antiapoptotic protein, B-cell lymphoma-2, at the mRNA and protein levels, similar to the effects of the positive control, selegiline. In contrast, paeoniflorin treatment downregulated the expression of pro-apoptotic proteins BCL2-Associated X2, α-synuclein, and PKC-δ at the mRNA and protein levels, as well as the level of the activated form of nuclear factor kappa B (p-NF-κB p65). Thus, our results showed that paeoniflorin exerts its neuroprotective effect by regulating the α-synuclein/PKC-δ signaling pathway to reduce neuronal apoptosis.