Schisandrin A alleviates renal fibrosis by inhibiting PKCß and oxidative stress.

BACKGROUND: Renal fibrosis is a common pathway that drives the advancement of numerous kidney maladies towards end-stage kidney disease (ESKD). Suppressing renal fibrosis holds paramount clinical importance in forestalling or retarding the transition of chronic kidney diseases (CKD) to renal failure. Schisandrin A (Sch A) possesses renoprotective effect in acute kidney injury (AKI), but its effects on renal fibrosis and underlying mechanism(s) have not been studied. STUDY DESIGN: Serum biochemical analysis, histological staining, and expression levels of related proteins were used to assess the effect of PKCß knockdown on renal fibrosis progression. Untargeted metabolomics was used to assess the effect of PKCß knockdown on serum metabolites. Unilateral Ureteral Obstruction (UUO) model and TGF-ß induced HK-2 cells and NIH-3T3 cells were used to evaluate the effect of Schisandrin A (Sch A) on renal fibrosis. PKCß overexpressed NIH-3T3 cells were used to verify the possible mechanism of Sch A. RESULTS: PKCß was upregulated in the UUO model. Knockdown of PKCß mitigated the progression of renal fibrosis by ameliorating perturbations in serum metabolites and curbing oxidative stress. Sch A alleviated renal fibrosis by downregulating the expression of PKCß in kidney. Treatment with Sch A significantly attenuated the upregulated proteins levels of FN, COL-I, PKCß, Vimentin and α-SMA in UUO mice. Moreover, Sch A exhibited a beneficial impact on markers associated with oxidative stress, including MDA, SOD, and GSH-Px. Overexpression of PKCß was found to counteract the renoprotective efficacy of Sch A in vitro. CONCLUSION: Sch A alleviates renal fibrosis by inhibiting PKCß and attenuating oxidative stress.

The store-operated Ca2+ channel Orai1α is required for agonist-evoked NF-κB activation by a mechanism dependent on PKCß2.

Store-operated Ca2+ entry is a ubiquitous mechanism for Ca2+ influx in mammalian cells that regulates a variety of physiological processes. The identification of two forms of Orai1, the predominant store-operated channel, Orai1α and Orai1ß, raises the question whether they differentially regulate cell function. Orai1α is the full-length Orai1, containing 301 amino acids, whereas Orai1ß lacks the N-terminal 63 amino acids. Here, using a combination of biochemistry and imaging combined with the use of human embryonic kidney 293 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1α plays a relevant role in agonist-induced NF-κB transcriptional activity. In contrast, functional Orai1ß is not required for the activation of these transcription factors. The role of Orai1α in the activation of NF-κB is entirely dependent on Ca2+ influx and involves PKCß activation. Our results indicate that Orai1α interacts with PKCß2 by a mechanism involving the Orai1α exclusive AKAP79 association region, which strongly suggests a role for AKAP79 in this process. These findings provide evidence of the role of Orai1α in agonist-induced NF-κB transcriptional activity and reveal functional differences between Orai1 variants.

Pharmacophore modeling, docking and molecular dynamics simulation for identification of novel human protein kinase C beta (PKCß) inhibitors.

Protein kinase Cß (PKCß) is considered as an attractive molecular target for the treatment of COVID-19-related acute respiratory distress syndrome (ARDS). Several classes of inhibitors have been already identified. In this article, we developed and validated ligand-based PKCß pharmacophore models based on the chemical structures of the known inhibitors. The most accurate pharmacophore model, which correctly predicted more than 70% active compounds of test set, included three aromatic pharmacophore features without vectors, one hydrogen bond acceptor pharmacophore feature, one hydrophobic pharmacophore feature and 158 excluded volumes. This pharmacophore model was used for virtual screening of compound collection in order to identify novel potent PKCß inhibitors. Also, molecular docking of compound collection was performed and 28 compounds which were selected simultaneously by two approaches as top-scored were proposed for further biological research. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02075-y.

PKCß Facilitates Leukemogenesis in Chronic Lymphocytic Leukaemia by Promoting Constitutive BCR-Mediated Signalling.

B cell antigen receptor (BCR) signalling competence is critical for the pathogenesis of chronic lymphocytic leukaemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKCα function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKCßII upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKCα-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKCα-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKCα-KR expressing cells, coincident with upregulation of PKCßII: supported by activation of key signalling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKCßII inhibitor) treatment of PKCα-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKCßII in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKCα-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKCß expression facilitates leukemogenesis and identify that BCR-mediated signalling is a key driver of CLL development in the PKCα-KR model.

MARCKS Is an Essential Regulator of Reactive Oxygen Species Production in the Monocytic Cell Type.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitous protein mediating versatile effects in a variety of cell types, including actin crosslinking, signal transduction, and intracellular transport processes. MARCKS's functional role in monocyte/macrophages, however, has not yet been adequately addressed. Thus, the aim of this study was to further elucidate the impact of MARCKS on central cellular functions of monocytic cells. To address this topic, we generated monocytic THP-1 (Tohoku Hospital Pediatrics-1)-derived MARCKS wildtype and knockout (KO) cells using the CRISPR/Cas9 technique. Remarkably, in the absence of MARCKS, both total and intracellular reactive oxygen species (ROS) production were strongly suppressed but restored following transient MARCKS re-transfection. In contrast, proliferation, differentiation, cytokine expression, and phagocytosis remained unaltered. A complete inhibition of ROS production could also be achieved in THP-1-derived PKCß KO cells or in PKC inhibitor Staurosporine-treated primary human monocytes. MARCKS deficiency also involved reduced basal Akt phosphorylation and delayed re-phosphorylation. Further analyses indicated that long-term TNF pre-incubation strongly enhances monocytic ROS production, which was completely blocked in MARCKS and PKCß KO cells. Collectively, our study demonstrates that MARCKS is an essential molecule enabling ROS production by monocytic cells and suggests that MARCKS is part of a signal cascade involved in ROS formation.

PKCß Inhibition Promotes TXNIP Degradation to Ameliorate Pancreatic ß-Cell Dysfunction.

INTRODUCTION: Pancreatic ß-cell dysfunction is largely regulated by TXNIP accumulation, we have previously disclosed the role of PKA in TXNIP degradation during ß-cell dysfunction. However, whether other kinases (PKCs) still regulate TXNIP is unclear, which is beneficial to alleviate ß-cell dysfunction. METHODS: Thapsigargin (ER stress inducer) was used to induce ß-cell dysfunction. PKC's inhibitors were screened by Western blotting indicated by TXNIP. Also RT-qPCR and Co-immunoprecipitation were applied for evaluating the ß-cell improvement ability of PKC's inhibitors, and the insulin secretion ability was evaluated by glucose-stimulated insulin secretion assay. RESULTS: PKC's pan-inhibitor, Ro31-8220, decreased ß-cell apoptosis and improved insulin secretion under ER stress or high glucose (HG) conditions. Further studies showed that Ro31-8220 reduced ER stress or HG-induced TXNIP levels. On the other side, PKCß activation or overexpression could reverse the effect of Ro31-8220 on TXNIP. Also, PKCß selective inhibitor, ruboxistaurin, induced TXNIP degradation as significantly as Ro31-8220 did. CONCLUSION: This study reveals the regulating mechanism of PKCß inhibitor on TXNIP degradation to improve ß-cell dysfunction. These data indicated PKCß inhibitor is a promising agent for ameliorating ß-cell dysfunction through TXNIP.

Parallel kinase pathways stimulate actin polymerization at depolarized mitochondria.

Mitochondrial damage (MtD) represents a dramatic change in cellular homeostasis, necessitating metabolic changes and stimulating mitophagy. One rapid response to MtD is a rapid peri-mitochondrial actin polymerization termed ADA (acute damage-induced actin). The activation mechanism for ADA is unknown. Here, we use mitochondrial depolarization or the complex I inhibitor metformin to induce ADA. We show that two parallel signaling pathways are required for ADA. In one pathway, increased cytosolic calcium in turn activates PKC-ß, Rac, WAVE regulatory complex, and Arp2/3 complex. In the other pathway, a drop in cellular ATP in turn activates AMPK (through LKB1), Cdc42, and FMNL formins. We also identify putative guanine nucleotide exchange factors for Rac and Cdc42, Trio and Fgd1, respectively, whose phosphorylation states increase upon mitochondrial depolarization and whose suppression inhibits ADA. The depolarization-induced calcium increase is dependent on the mitochondrial sodium-calcium exchanger NCLX, suggesting initial mitochondrial calcium efflux. We also show that ADA inhibition results in enhanced mitochondrial shape changes upon mitochondrial depolarization, suggesting that ADA inhibits these shape changes. These depolarization-induced shape changes are not fragmentation but a circularization of the inner mitochondrial membrane, which is dependent on the inner mitochondrial membrane protease Oma1. ADA inhibition increases the proteolytic processing of an Oma1 substrate, the dynamin GTPase Opa1. These results show that ADA requires the combined action of the Arp2/3 complex and formin proteins to polymerize a network of actin filaments around mitochondria and that the ADA network inhibits the rapid mitochondrial shape changes that occur upon mitochondrial depolarization.

[Acupuncture intervention induced improvement of oxidative stress by regulating PKCß/P66shc signaling in obese diabetic rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36)-"Sanyinjiao"(SP6) on glucose and lipid metabolism and insulin resistance (IR) in obese diabetic rats, so as to explore its mechanism underlying improvement of obesity diabetes. METHODS: SPF male rats were randomly divided into normal control, model, meridian-acupoint EA (acupoint), non-meridian non-acupoint EA (non-acupoint), and medication (metformin) groups, with 10 rats in each group. The diabetes model was established by feeding the rats with high-fat diet for 8 weeks. EA (1.5 mA, 10 Hz/100 Hz) was applied to unilateral ST36 and SP6 for 20 min, once daily (except Sundays) for 4 weeks. Rats of the medication group were treated by gavage of metformin (300 mg/kg) once daily for 4 weeks (except Sundays). The body weight and length were measured and the Lee's index was calculated. The contents of total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C) in the plasma were detected by using a full-automatic biochemical analyzer. The content of fasting serum insulin (FINS) was assayed by using radioimmunoassay, the fasting blood glucose (FBG) was measured, and serum superoxide dismutase (SOD) activity by using xanthine oxidase method, serum malondialdehyde (MDA) by color method, serum glutathione peroxidase (GSH-Px) activity by indirect method, reactive oxygen species (ROS) by Dithio-bis-nitrobenzoic acid (DTNB) direct method, and the homeostasis model assessment of IR (HOMA-IR) and insulin sensitive index (ISI) were calculated. The expression levels of pancreatic tissue P66shc mRNA and PKCß mRNA were detected by using RT-PCR, and the histopathological changes of the liver and adipose tissues were observed after H.E. staining. RESULTS: Compared with the normal control group, the Lee's index, levels of FBG, FINS, HOMA-IR, TC, TG, LDL-C, MDA, ROS, and P66shc mRNA and PKCß mRNA expressions were significantly increased (P<0.05，P<0.01), and ISI, HDL-C, SOD, GSH-Px significantly decreased (P<0.05, P<0.01) in the model group. After the interventions, the levels of Lee's index,levels of FBG, FINS, HOMA-IR, TC, TG, LDL-C, MDA, ROS, and expressions of P66shc mRNA and PKCß mRNA were remarkably down-regulated (P<0.05, P<0.01), and those of ISI, HDL-C, SOD, and GSH-Px up-regulated (P<0.05, P<0.01) in both EA and medication groups. H.E. staining showed many white adipocytes in the adipose tissue, radial and cord-like arrangement of liver cells, and many of them with vacuoles in the cytoplasm of small vesicular lipid droplets in the model group; and relative reduction of white adipocytes in number, smaller in cell body, and no obvious abnormal changes of structure and arrangement of liver cells in the EA and medication groups. CONCLUSION: EA of ST36 and SP6 can improve glucose and lipid metabolism and IR in obese diabetic rats, which may be related to its function in suppressing PKCß/P66shc signaling and oxidative stress.

P2X7 Receptor Deficiency Ameliorates STZ-induced Cardiac Damage and Remodeling Through PKCß and ERK.

Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus which result in cardiac remodeling and subsequent heart failure. However, the role of P2X7 receptor (P2X7R) in DCM has yet to be elucidated. The principal objective of this study was to investigate whether P2X7R participates in the pathogenesis of DCM. In this study, the C57BL/6 diabetic mouse model was treated with a P2X7R inhibitor (A438079). Cardiac dysfunction and remodeling were attenuated by the intraperitoneal injection of A438079 or P2X7R deficiency. In vitro, A438079 reduced high glucose (HG) induced cell damage in H9c2 cells and primary rat cardiomyocytes. Furthermore, HG/streptozotocin (STZ)-induced P2X7R activation mediated downstream protein kinase C-ß (PKCß) and extracellular regulated protein kinases (ERK) activation. This study provided evidence that P2X7R plays an important role in the pathogenesis of STZ-induced diabetic cardiac damage and remodeling through the PKCß/ERK axis and suggested that P2X7R might be a potential target in the treatment of diabetic cardiomyopathy.

NAC antagonizes arsenic-induced neurotoxicity through TMEM179 by inhibiting oxidative stress in Oli-neu cells.

Arsenic is one of the most common environmental pollutants. Neurotoxicity induced by arsenic has become a major public health concern. However, the effects of arsenic-induced neurotoxicity in the brain and the underlying molecular mechanisms are not well understood. N-acetyl-cysteine (NAC) is a thiol-based antioxidant that can antagonize heavy metal-induced neurotoxicity by scavenging reactive oxygen species (ROS). Here, we used the mouse oligodendrocyte precursor cell (OPC) line Oli-neu to explore the neurotoxic effects of arsenic and the protective effects of NAC. We found that arsenic exposure decreased cell viability, increased oxidative stress, caused mitochondrial dysfunction, and led to apoptosis of Oli-neu cells. Furthermore, we revealed that NAC treatment reversed these neurotoxic effects of arsenic. TMEM179, a key membrane protein, was found highly expressed in OPCs and to be an important factor in maintaining mitochondrial functions. We found that TMEM179 played a critical role in mediating the neurotoxic effects of arsenic and the protective role of NAC. PKCß is a downstream factor through which TMEM179 regulates the expression of apoptosis-related proteins. This study improves our understanding of the neurotoxic effects and mechanisms of arsenic exposure and the protective effects of NAC. It also identifies a potential molecular target, TMEM179, for the treatment of arsenic-induced neurotoxicity.

Selective inhibition of PKCß2 improves Caveolin-3/eNOS signaling and attenuates lipopolysaccharide-induced injury by inhibiting autophagy in H9C2 cardiomyocytes.

Lipopolysaccharide (LPS)-induced autophagy is involved in sepsis-associated myocardial injury with increased PKCß2 activation. We previously found hyperglycemia-induced PKCß2 activation impaired the expression of caveolin-3 (Cav-3), the dominant isoform to form cardiomyocytes caveolae which modulate eNOS signaling to confer cardioprotection in diabetes. However, little is known about the roles of PKCß2 in autophagy and Cav-3/eNOS signaling in cardiomyocytes during LPS exposure. We hypothesize LPS-induced PKCß2 activation promotes autophagy and impairs Cav-3/eNOS signaling in LPS-treated cardiomyocytes. H9C2 cardiomyocytes were treated with LPS (10 µg/mL) in the presence or absence of PKCß2 inhibitor CGP53353 (CGP, 1 µM) or autophagy inhibitor 3-methyladenine (3-MA, 10 µM). LPS stimulation induced cytotoxicity overtime in H9C2 cardiomyocytes, accompanied with excessive PKCß2 activation. Selective inhibition of PKCß2 with CGP significantly reduced LPS-induced cytotoxicity and autophagy (measured by LC-3II, Beclin-1, p62 and autophagic flux). In addition, CGP significantly attenuated LPS-induced oxidative injury, and improved Cav-3 expression and eNOS activation, similar effects were shown by the treatment of autophagy inhibitor 3-MA. LPS-induced myocardial injury is associated with excessive PKCß2 activation, which contributes to elevated autophagy and impaired Cav-3/eNOS signaling. Selective inhibition of PKCß2 improves Cav-3/eNOS signaling and attenuates LPS-induced injury through inhibiting autophagy in H9C2 cardiomyocytes.

Immune modulatory effects of Idelalisib in stromal cells of chronic lymphocytic leukemia.

Molecular targets of tyrosine kinase inhibitors are not restricted to the B-cell compartment but also regulate functions in the tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases like protein kinase C (PKC)-ß is essential for the formation of a microenvironment supporting leukemic growth. Here we describe the effect of Idelalisib on the PKCß/NF-κB and Notch pathway in stromal cells upon contact to primary chronic lymphocytic leukemia cells (CLL). There is no Idelalisib-dependent regulation of the Notch expression in stromal cells, whereas Idelalisib induces PKCß expression and activates the canonical NF-κB pathway. Idelalisib deregulates important immune-modulatory proteins in activated stromal cells, which might provoke the patient's side effects. Additionally, we established a 3D-stroma/leukemia model, that can give us a more defined look into the communication between tumor and stromal cells than standard cell cultures. This opens up the possibility to improve therapies, especially in the context of minimal-residual disease.

PKC-ß/Alox5 axis activation promotes Bcr-Abl-independent TKI-resistance in chronic myeloid leukemia.

Bcr-Abl independent resistance to tyrosine kinase inhibitor (TKI) is a crucial factor lead to relapse or acute leukemia transformation in chronic myeloid leukemia (CML). However, its mechanism is still unclear. Herein, we found that of nine common protein kinases C (PKCs), PKC-ß overexpression was significantly related with TKI resistance. Blockage of its expression in CD34+ cells and CML cell lines increased sensitivity to imatinib. Then, eighty-four leukemia related genes were compared between TKI-resistant CML cell lines with PKC-ß silenced or not. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that Arachidonate 5-lipoxygenase (Alox5) and its relative pathway mainly participated in the resistance induced by PKC-ß overexpression. It's also observed that Alox5 was increased not only in bone marrow biopsy but also in CD34+ cells derived from IM-resistant CML patients. The signaling pathway exploration indicated that ERK1/2 pathway mediates Alox5 upregulation by PKC-ß. Meanwhile, we also proved that Alox5 induces TKI-insensitivity in CML through inactivation of PTEN. In vivo experiment, PKC-ß elective inhibitor LY333531 prolonged survival time in CML-PDX mice model. In conclusion, targeted on PKC-ß overexpression might be a novel therapy mechanism to overcome TKI-resistance in CML.

Protein kinase C-ß-dependent changes in the glucose metabolism of bone marrow stromal cells of chronic lymphocytic leukemia.

Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-ß (PKCß) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCß on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCß that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCß results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCß-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCß and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.

High glucose-induced effects on Na+-K+-2Cl- cotransport and Na+/H+ exchange of blood-brain barrier endothelial cells: involvement of SGK1, PKCßII, and SPAK/OSR1.

Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.

PKCß increases ROS levels leading to vascular endothelial injury in diabetic foot ulcers.

OBJECTIVE: To explore the role and mechanism of oxidative stress injury in the diabetic foot. METHODS: Immunohistochemistry and staining were used to detect changes in diabetic foot tissue, and the CCK-8 method was used to measure high glucose effect on cell viability. The DCFH-DA assay was used to detect the intracellular ROS content, and colorimetric methods were used to detect the activities of the CAT and SOD enzymes and the NO and MDA content in tissues and cells. In addition, the protein expression levels of PKCß, p66shc, eNOS, ICAM-1 and NF-κB in tissues and cells were detected by Western blotting, and the distribution of p66shc and eNOS was observed by immunofluorescence. RESULTS: The results of clinical specimens experiments showed that the DFU group exhibited disordered morphology and increased glucose metabolism, decreased activities of the enzymes CAT and SOD in tissues, and increased MDA and NO contents compared to those in the CON group. Furthermore, protein levels of the p-PKCß, p-p66shc, ICAM-1, and p-NF-κB were increased, and eNOS protein level was decreased; these results were consistent in clinical specimens and in vitro experiments. CONCLUSIONS: High glucose levels may induce oxidative stress injury in cells and tissues by activating the PKCß-p66shc signaling pathway.

PKCß/NF-κB pathway in diabetic atrial remodeling.

Atrial remodeling in diabetes is partially attributed to NF-κB/TGF-ß signal transduction pathway activation. We examined whether the hyperglycemia-induced increased expression of NF-κB/TGF-ß was dependent upon protein kinase C-ß (PKCß) and tested the hypothesis that selective inhibition of PKCß using ruboxistaurin (RBX) can reduce NF-κB/TGF-ß expression and inhibit abnormal atrial remodeling in streptozotocin (STZ)-induced diabetic rats. The effects of PKCß inhibition on NF-κB/TGF-ß signal transduction pathway-mediated atrial remodeling were investigated in STZ-induced diabetic rats. Mouse atrial cardiomyocytes (HL-1 cells) were cultured in low- or high-glucose or mannitol conditions in the presence or absence of small interference RNA that targeted PKCß. PKCß inhibition using ruboxistaurin (RBX, 1 mg/kg/day) decreased the expression of NF-κBp65, p-IκB, P38MARK, TNF-α, TGF-ß, Cav1.2, and NCX proteins and inducibility of atrial fibrillation (AF) in STZ-induced diabetic rats. Exposure of cardiomyocytes to high-glucose condition activated PKCß and increased NF-κB/TGF-ß expression. Suppression of PKCß expression by small interference RNA decreased high-glucose-induced NF-κB and extracellular signal-related kinase activation in HL-1 cells. Pharmacological inhibition of PKCß is an effective method to reduce AF incidence in diabetic rat models by preventing NF-κB/TGF-ß-mediated atrial remodeling.

Inhibiting PKCß2 protects HK-2 cells against meglumine diatrizoate and AGEs-induced apoptosis and autophagy.

BACKGROUND: Contrast induced diabetic nephropathy (CIN) is an important cause of hospital-acquired acute renal failure. Our aim was to observe the effect of protein kinase C ß2 (PKCß2) knockdown on human proximal tubular epithelial cells (HK-2 cells) against meglumine diatrizoate and advanced glycation end products (AGEs)-induced apoptosis and autophagy. METHODS: Cell viability was detected using cell counting kit-8 (CCK-8) assay in HK-2 cells after disposal with meglumine diatrizoate and AGEs with or without PKCß2 siRNA/inhibitor LY333531. Flow cytometry and western blot were used to test cell apoptosis and the related protein levels in meglumine diatrizoate and AGEs co-treated HK-2 cells with or without PKCß2 siRNA/inhibitor LY333531. Autophagy related proteins were detected using western blot. Immunofluorescence staining was used to examine the autophagy-specific protein light chain 3 (LC3), and autophagosome and autolysosome formation was observed under a transmission electron microscopy. RESULTS: CCK-8 assay results showed that meglumine diatrizoate inhibited AGEs-induced HK-2 cell viability. Furthermore, meglumine diatrizoate promoted cell apoptosis and the expression level of caspase3 in AGEs-induced HK-2. Western blot results showed that meglumine diatrizoate elevated the expression levels of PKCß2 and p-PKCß2 in AGEs-induced HK-2 cells, and up-regulated the expression level of Beclin-1 and the ratio of LC3 II/LC3 I, and down-regulated the expression level of p62 in AGEs-induced HK-2 cells. We found that PKCß2 knockdown alleviated meglumine diatrizoate and AGEs-induced HK-2 cell apoptosis and autophagy. Intriguingly, PKCß2 inhibitor LY333531 reversed 3-methyladenine (3-MA)-induced autophagy inhibition in meglumine diatrizoate and AGEs-induced HK-2 cells. CONCLUSIONS: Our findings reveal that inhibiting PKCß2 protects HK-2 cells against meglumine diatrizoate and AGEs-induced apoptosis and autophagy, which provide a novel therapeutic insight for CIN in diabetic patients.

Chronic morphine regulates TRPM8 channels via MOR-PKCß signaling.

Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCß), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCß-mediated reduction of TRPM8 desensitization. This MOR-PKCß dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCß as important targets for opioid-induced cold hypersensitivity.

PKCß and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats.

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCß and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCß and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCß inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCß-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCß in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCß activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCß and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.