Protein Kinase CβII Research Articles

An emerging role of ferulic acid on sub-adult grass carp (Ctenopharyngodon idellus): Increasing protein digestion and regulating amino acid and oligopeptide transporters expression

This study was conducted to evaluate the effect of supplement ferulic acid on growth performance, digestive ability and transport function of amino acids and oligopeptides in the intestine of sub-adult grass carp (Ctenopharyngodon idellus). The experiment was divided into two parts: a. Digestion experiment: healthy grass carp (678.83±1.00 g) were divided into two groups: control group (0 mg/kg FA) and ferulic acid group (100 mg/kg FA) for 2 weeks; b. Growth experiment: healthy grass carp (678.83±1.00 g) were divided into five treatment groups, the control group (FA0) with no ferulic acid and the experimental group supplemented with ferulic acid 50 (FA50), 100 (FA100), 150 (FA150) and 200 (FA200) mg/kg feed for 9 weeks. The results showed that a. Ferulic acid supplementation increased the apparent digestibility of crude protein and crude fat in grass carp. b. Ferulic acid supplementation improved the growth performance and feed efficiency (FE) of grass carp; increased the activities of trypsin and Na+/K+-ATPase; and up-regulated the levels of mRNAs of some neutral and cationic (non-anionic) amino acid transporter proteins (Solute Carrier Family7member1 (SLC7A7), SLC7A8, etc.) and H+/oligopeptide transporter protein 1 (PepT1) gene and protein levels. In addition, ferulic acid promoted the gene expression levels of protein kinase B (Akt), target of rapamycin (TOR); up-regulated the mRNA levels of tail-side-associated homologous frame transcription factor 2 (Cdx2), cAMP response element-binding protein (CREB), and transcriptional co-activator-specific protein 1 (Sp1), and down-regulated the gene and protein levels of protein kinase CβII (PKCβII). In a word, dietary ferulic acid promoted growth performance, digestion, as well as amino acid and oligopeptide transport capacities. Finally, based on regression analyses of percentage weight gain (PWG), FE, specific growth rate (SGR), intestine fold height, trypsin activity, and Na+/K+-ATPase activity, we recommend optimal supplementation levels of ferulic acid in sub-adult grass carp of 92.18–120.60 mg/kg diet.

Reducing the Incidence of Post – Myocardial Infarction Heart Failure in an Era of Improved Primary Percutaneous Coronary Intervention through Novel Therapeutics (Protein Kinase C Beta II Inhibitor)

Heart failure (HF) complicating myocardial infarction (MI) remains a major source of morbidity, mortality and a huge health-care burden in the world. The incidence of symptomatic HF after primary percutaneous coronary intervention (PPCI) has been widely reported. PPCI has improved early survival of MI, but its impact on downstream HF is deliberated. Infarct size is the major determinant of the development of HF and prognosis. Efforts to reduce the size will therefore have a significant impact on the incidence of HF after PPCI. Resuscitation of the ischemic myocardium, heavily rests on timely reperfusion. However, cardiomyocyte death still occurs due to ischemic/reperfusion (I/R) injury mediated by oxidative stress. This phenomenon is known to largely contribute to the size of infarct. Activation by protein kinase C-βII (PKCβII) is attributed to the generation of reactive oxygen species (ROS) during this process and inhibition of this drive could salvage the myocardial tissue and subsequently preserve cardiac function. We hypothesized that myristic acid (Myr) and trans-activator of transcription (Tat) conjugated PKCβII inhibitor (Myr-Tat-PKCβII-) will exhibit robust cardioprotective effects in an in vivo porcine model, reflective of our ex vivo rat hearts model. Male Yorkshire pigs underwent regional I/R by occluding the second branch of the left anterior descending artery (LAD). This was achieved in the catheterization laboratory guided by fluoroscopy. A bolus of Myr-Tat-PKCβII- or scrambled control was infused into the LAD during reperfusion. Cardiac function was evaluated by an echocardiogram during the entire procedure, and assessed as the relative change at the end of 3hr reperfusion compared to baseline. Post-reperfused hearts were stained with Evans Blue dye to identify the area a risk and 1% triphenyltetrazolium chloride to delineate the area of necrosis. Infarct size and ejection fraction (EF) were analyzed via Student’s t- test. From mean baseline EF (61.4±0.5%), Myr-Tat-PKCβII- preserved EF with a relative change of 1.2±2.8% compared to 8.9±2.2% in control hearts (p<0.05) at the end of reperfusion. Myr-Tat-PKCβII- reduced infarct size to 13.5±3.9% (n=4) compared to scrambled control hearts (27.5 ±7.9%, n=6). These results suggest Myr-Tat-PKCβII- significantly preserve EF after PPCI and reduce infarct size when given at reperfusion onset. This study gives a solid premise for future examination of Myr-Tat-PKCβII- in an 8 week in-vivo, porcine MI survival study.

Identification of novel protein kinase C-βII inhibitors: virtual screening, molecular docking and molecular dynamics simulation studies.

Diabetic wounds (DWs) are the major end-stage manifestation encountered in diabetic patients. The two major pathways involved in the pathogenesis of DW are impaired angiogenesis and unnecessary NETosis, which are regulated by a common enzyme called protein kinase C (PKC)-βII. PKC-βII is a conventional isoform of PKC family that can be activated by calcium and diacylglycerol. PKC-βII possesses a specific expression profile and plays a distinct role in various cellular and molecular functions. The pathogenic role of PKC-βII and its involvement in the impairment of wound healing suggested that PKC-βII plays a potential role in DW progression. Hence, there is a renewed interest in developing specific inhibitors of PKC-βII. In the present study, receptor-based virtual screening was performed for the identification of potential PKC-βII inhibitors using TimTec, Enamine, Zinc and Specs databases. A total of 595 candidate compounds were evaluated based on absorption, distribution, metabolism, excretion and toxicity, standard precision docking. Further, extra-precision docking and binding free energy calculations were carried out for top-ranked compounds. Based on Glide score and protein-ligand interactions, we have identified compound 1 as a potential inhibitor. Finally, molecular dynamics (MD) simulation was performed for top compound 1 using the Desmond module (Schrödinger suite) to identify the structural stability of the protein-ligand complex. Gratifyingly, MD trajectory analysis demonstrated the stable binding conformation of compound 1 with PKC-βII enzyme. In silico approaches incorporated in this study provide a set of new putative PKC-βII inhibitors which could be potential leads to develop DW therapeutics.

Oxidative Stress in Intestinal Ischemia-Reperfusion.

Ischemia-reperfusion (I/R) injury is a manifestation of tissue or organ damage that is followed by ischemia and exacerbated by the return of blood flow to a previously damaged tissue or organ. The intestines are one of the most sensitive tissues and organs to I/R injury. Moreover, the adverse consequences of intestinal I/R (II/R) injury are not limited to the intestine itself and can also lead to damage of the distant tissues and organs. The mechanism of II/R is extremely complex and oxidative stress is the key link in the pathogenesis of II/R injury. This study summarizes the roles of oxidative stress and its signaling pathways involved in II/R. The signaling pathways that mitigate II/R injury include the nuclear factor erythroid-related factor 2 (Nrf2)-mediated signaling pathway, Wnt/β-catenin pathway, and phosphatidylinositol kinase 3 (PI3K)/Akt pathway; those that aggravate II/R injury include the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, Toll-like receptor (TLR) receptor-mediated signaling pathway, protein kinase CβII (PKCβII)/p66shc pathway, and microRNA (miRNA)/p66shc pathway; the effect of miRNA on related pathways and mitochondrial DNA translocation. The aforementioned pathways provide new ideas for further exploring the occurrence and development of II/R and more effective treatments for II/R injury.

Nuclear lipidome is altered in amyotrophic lateral sclerosis: A pilot study.

Nucleocytosolic transport, a membrane process, is impaired in motor neurons in amyotrophic lateral sclerosis (ALS). This study analyzes the nuclear lipidome in motor neurons in ALS and examines molecular pathways linked to the major lipid alterations. Nuclei were obtained from the frozen anterior horn of the lumbar spinal cord of ALS patients and age-matched controls. Lipidomic profiles of this subcellular fraction were obtained using liquid chromatography and mass spectrometry. We validated the mechanisms behind presumable lipidomic changes by exploring ALS surrogate models including human motor neurons (derived from ALS lines and controls) subjected to oxidative stress, the hSOD-G93A transgenic mice, and samples from an independent cohort of ALS patients. Among the differential lipid species, we noted 41 potential identities, mostly belonging to phospholipids (particularly ether phospholipids, as plasmalogens), as well as diacylglycerols and triacylglycerides. Decreased expression of alkyldihydroxyacetonephosphate synthase (AGPS)-a critical peroxisomal enzyme in plasmalogen synthesis-is found in motor neuron disease models; this occurs in parallel with an increase in the expression of sterol carrier protein 2 (SCP2) mRNA in ALS and Scp2 levels in G93A transgenic mice. Further, we identified diminished expression of diacylglycerol-related enzymes, such as phospholipase C βI (PLCβI) and protein kinase CβII (PKCβII), linked to diacylglycerol metabolism. Finally, lipid droplets were recognized in the nuclei, supporting the identification of triacylglycerides as differential lipids. Our results point to the potentially pathogenic role of altered composition of nuclear membrane lipids and lipids in the nucleoplasm in the anterior horn of the spinal cord in ALS. Overall, these data support the usefulness of subcellular lipidomics applied to neurodegenerative diseases.

Glutathione Peroxidase-1 Knockout Facilitates Memory Impairment Induced by β-Amyloid (1-42) in Mice via Inhibition of PKC βII-Mediated ERK Signaling; Application with Glutathione Peroxidase-1 Gene-Encoded Adenovirus Vector.

A growing body evidence suggests that selenium (Se) deficiency is associated with an increased risk of developing Alzheimer's disease (AD). Se-dependent glutathione peroxidase-1 (GPx-1) of a major antioxidant enzyme, and the most abundant isoform of GPx in the brain. In the present study, we investigated whether GPx-1 is protective against memory impairments induced by beta-amyloid (Aβ) (1-42) in mice. As the alteration of protein kinase C (PKC)-mediated ERK activation was recognized in the early stage of AD, we examined whether the GPx-1 gene modulates Aβ (1-42)-induced changes in PKC and ERK levels. We observed that Aβ (1-42) treatment (400pmol, i.c.v.) significantly decreased PKC βII expression in the hippocampus of mice. Aβ (1-42)-induced neurotoxic changes [i.e., oxidative stress (i.e., reactive oxygen species, 4-hydroxy-2-noneal, and protein carbonyl), reduced PKC βII and phospho-ERK expressions, and memory impairment under Y-maze and passive avoidance test] were more pronounced in GPx-1 knockout than in wild type mice. Importantly, exposure to a GPx-1 gene-encoded adenovirus vector (Adv-GPx-1) significantly increased GPx-1 mRNA and GPx activity in the hippocampus of GPx-1 knockout mice. Adv-GPx-1 exposure also significantly blocked the neurotoxic changes induced by Aβ (1-42) in GPx-1 knockout mice. Treatment with ERK inhibitor U0126 did not significantly change Adv-GPx-1-mediated attenuation in PKC βII expression. In contrast, treatment with PKC inhibitor chelerythrine (CHE) reversed Adv-GPx-1-mediated attenuation in ERK phosphorylation, suggesting that PKC βII-mediated ERK signaling is important for Adv-GPx-1-mediated potentials against Aβ (1-42) insult. Our results suggest that treatment with the antioxidant gene GPx-1 rescues Aβ (1-42)-induced memory impairment via activating PKC βII-mediated ERK signaling.

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCβ/p66Shc axis and activation of FOXO-3a

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p66Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- β-1 (TGF-β1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CβII (PKCβII), NADPH oxidase, and p66Shc, and decreased the activation of p66Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCβII/p66Shc axis.

A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CβII

Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α–converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA–mediated knockdown, and overexpression of dominant-negative PKCβII variants indicated that PKCβII is a major player in JNK inhibition– and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCβII and tumor necrosis factor-α–converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCβII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.

Metabotropic signaling cascade involved in α4β2 nicotinic acetylcholine receptor-mediated PKCβII activation

Nicotinic acetylcholine receptors (nAChRs) belong to the ionophore receptor family, which regulates plasma membrane conductance to Na+, K+, and Ca2+ ions. Some studies, however, have shown that nAChRs also employ second messengers for intracellular signaling. We previously showed that α4β2 nAChR mediates the translocation of protein kinase CβII (PKCβII) from the cytoplasm to the plasma membrane, which is a typical activation marker for PKCβII. In this study, we investigated the molecular mechanisms underlying PKCβII activation through α4β2 nAChR. α4β2 nAChR is the most abundant nAChR subtype and is implicated in various brain functions and diseases. Putative α4β2 nAChR signaling components were identified by knockdown or chemical inhibition of candidate proteins, and the signaling cascade was deduced by protein interactions in predicted cellular components. α4β2 nAChR-mediated PKCβII translocation was found to occur in an ionophore activity-independent manner. Nicotinic stimulation of α4β2 nAChR activated Src in a β-arrestin1 and 14–3-3η-dependent manner. Activated Src phosphorylated the tyrosine residue(s) on Syk molecules, which in turn interacted with phospholipase C γ1 to trigger the translocation of PKCβII to the cell membrane by elevating cellular diacylglycerol levels. The activated PKCβII in turn exerted a positive feedback effect on Src activation, suggesting that α4β2 nAChR signaling is amplified by a positive feedback loop. These findings provide novel information for unveiling the previously unclear metabotropic second messenger-based signal transduction pathway of nAChRs.

Hyperglycaemia cause vascular inflammation through advanced glycation end products/early growth response-1 axis in gestational diabetes mellitus.

Hyperglycaemia during pregnancy is the main reason for developing diabetes mediated vascular complications. Advanced glycation end products (AGEs) are formed due to non-enzymatic glycation of proteins, lipids and nucleic acids during hyperglycaemia. It has the potential to damage vasculature by modifying the substrate or by means of AGEs and receptor of AGE (RAGE) interaction. It has been linked with the pathogenesis of various vascular diseases including coronary heart disease, atherosclerosis, restenosis etc. This study was carried out to investigate the role of AGEs-EGR-1 pathway in gestational diabetes mellitus (GDM) vascular inflammation. Human umbilical vein endothelial cells(HuVECs) isolated from normal glucose tolerant mothers were subjected to various treatments including high glucose, silencing of early growth response (EGR)-1, blockade of protein kinase C (PKC) β, blocking extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and treatment with AGEs and assayed for EGR-1, tissue factor (TF) and soluble intercellular adhesion molecule (sICAM)-1. Similarly, umbilical veinendothelial cells isolated from normal and GDM mothers were assayed for EGR-1, TF, and sICAM-1. There was a significant increase in EGR-1 and TF levels in HuVECs isolated form GDM mother's umbilical cord and normal HuVECs treated with high glucose condition. This was accompanied by elevated levels of sICAM-1 in high glucose treated cells. Our results revealed AGE-mediated activation of EGR-1 and its downstream genes via PKC βII and ERK1/2 signaling pathway. The present study demonstrated a novel mechanism of AGEs/ PKC βII/ ERK1/2/EGR-1 pathway in inducing vascular inflammation in GDM.

Selenoprotein S protects against high glucose-induced vascular endothelial apoptosis through the PKCβII/JNK/Bcl-2 pathway.

Vascular endothelial apoptosis is closely associated with the pathogenesis and progression of diabetic macrovascular diseases. Selenoprotein S (SelS) participates in the protection of vascular endothelial and smooth muscle cells from oxidative and endoplasmic reticulum stress-induced injury. However, whether SelS can protect vascular endothelium from high glucose (HG)-induced apoptosis and the underlying mechanism remains unclear. The present study preliminarily analyzed aortic endothelial apoptosis and SelS expression in diabetic rats in vivo and the effects of HG on human umbilical vein endothelial cell (HUVEC) apoptosis and SelS expression in vitro. Subsequently, SelS expression was up- or downregulated in HUVECs using the pcDNA3.1-SelS recombinant plasmid and SelS-specific small interfering RNAs, and the effects of high/low SelS expression on HG-induced HUVEC apoptosis and a possible molecular mechanism were analyzed. As expected, HG induced vascular endothelial apoptosis and upregulated endothelial SelS expression in vivo and in vitro. SelS overexpression in HUVECs suppressed HG-induced increase in apoptosis and cleaved caspase3 level, accompanied by reduced protein kinase CβII (PKCβII), c-JUN N-terminal kinase (JNK), and B-cell lymphoma/leukemia-2 (Bcl-2) phosphorylation. In contrast, inhibiting SelS expression in HUVECs further aggravated HG-induced increase in apoptosis and cleaved caspase3 level, which was accompanied by increased PKCβII, JNK, and Bcl-2 phosphorylation. Pretreatment with PKC activators blocked the protective effects of SelS and increased the apoptosis and cleaved caspase3 level in HUVECs. In summary, SelS protects vascular endothelium from HG-induced apoptosis, and this was achieved through the inhibition of PKCβII/JNK/Bcl-2 pathway to eventually inhibit caspase3 activation. SelS may be a promising target for the prevention and treatment of diabetic macrovascular complications.

Possible Role of Phosphatidylglycerol-Activated Protein Kinase C-βII in Keratinocyte Differentiation.

Background:The epidermis is a continuously regenerating tissue maintained by a balance between proliferation and differentiation, with imbalances resulting in skin disease. We have previously found that in mouse keratinocytes, the lipid-metabolizing enzyme phospholipase D2 (PLD2) is associated with the aquaglyceroporin, aquaporin 3 (AQP3), an efficient transporter of glycerol. Our results also show that the functional interaction of AQP3 and PLD2 results in increased levels of phosphatidylglycerol (PG) in response to an elevated extracellular calcium level, which triggers keratinocyte differentiation. Indeed, we showed that directly applying PG can promote keratinocyte differentiation.Objective:We hypothesized that the differentiative effects of this PLD2/AQP3/PG signaling cascade, in which AQP3 mediates the transport of glycerol into keratinocytes followed by its PLD2-catalyzed conversion to PG, are mediated by protein kinase CβII (PKCβII), which contains a PG-binding domain in its carboxy-terminus. Method: To test this hypothesis we used quantitative RT-PCR, western blotting and immunocytochemistry.Results:We first verified the presence of PKCβII mRNA and protein in mouse keratinocytes. Next, we found that autophosphorylated (activated) PKCβII was redistributed upon treatment of keratinocytes with PG. In the unstimulated state phosphoPKCβII was found in the cytosol and perinuclear area; treatment with PG resulted in enhanced phosphoPKCβII localization in the perinuclear area. PG also induced translocation of phosphoPKCβII to the plasma membrane. In addition, we observed that overexpression of PKCβII enhanced calcium- and PG-induced keratinocyte differentiation without affecting calcium-inhibited keratinocyte proliferation.Conclusion:These results suggest that the PG produced by the PLD2/AQP3 signaling module may function by activating PKCβII.

Stimulatory and inhibitory effects of PKC isozymes are mediated by serine/threonine PKC sites of the Cav2.3α1 subunits

Protein kinase C (PKC) isozymes modulate voltage-gated calcium (Cav) currents through Cav2.2 and Cav2.3 channels by targeting serine/threonine (Ser/Thr) phosphorylation sites of Cavα1 subunits. Stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) sites were identified in the Cav2.2α1 subunits to PKCs βII and ε. In the current study, we investigated if the homologous sites of Cav2.3α1 subunits (stimulatory: Thr-365, Ser-1995 and Ser-2011; inhibitory: Ser-369) behaved in similar manner. Several Ala and Asp mutants were constructed in Cav2.3α1 subunits in such a way that the Ser/Thr sites can be examined in isolation. These mutants or WT Cav2.3α1 along with auxiliary β1b and α2/δ subunits were expressed in Xenopus oocytes and the effects of PKCs βII and ε studied on the barium current (IBa). Among these sites, stimulatory Thr-365 and Ser-1995 and inhibitory Ser-369 behaved similar to their homologs in Cav2.2α1 subunits. Furthermore PKCs produced neither stimulation nor inhibition when stimulatory Thr-365 or Ser-1995 and inhibitory Ser-369 were present together. However, the PKCs potentiated the IBa when two stimulatory sites, Thr-365 and Ser-1995 were present together, thus overcoming the inhibitory effect of Ser-369. Taken together net PKC effect may be the difference between the responses of the stimulatory and inhibitory sites.

Transcriptional mechanism of vascular endothelial growth factor-induced expression of protein kinase C\u03b2II in chronic lymphocytic leukaemia cells

A key feature of chronic lymphocytic leukaemia (CLL) cells is overexpressed protein kinase CβII (PKCβII), an S/T kinase important in the pathogenesis of this and other B cell malignancies. The mechanisms contributing to enhanced transcription of the gene coding for PKCβII, PRKCB, in CLL cells remain poorly described, but could be important because of potential insight into how the phenotype of these cells is regulated. Here, we show that SP1 is the major driver of PKCβII expression in CLL cells where enhanced association of this transcription factor with the PRKCB promoter is likely because of the presence of histone marks permissive of gene activation. We also show how vascular endothelial growth factor (VEGF) regulates PRKCB promoter function in CLL cells, stimulating PKCβ gene transcription via increased association of SP1 and decreased association of STAT3. Taken together, these results are the first to demonstrate a clear role for SP1 in the up regulation of PKCβII expression in CLL cells, and the first to link SP1 with the pathogenesis of this and potentially other B cell malignancies where PKCβII is overexpressed.

Insights into length-dependent regulation of cardiac cross-bridge cycling kinetics in human myocardium

Cross-bridge cycling kinetics play an essential role in the heart's ability to contract and relax. The rate of tension redevelopment (ktr) slows down as a muscle length is increased in intact human myocardium. We set out to determine the effect of rapid length step changes and protein kinase A (PKA) and protein kinase C-βII (PKC-βII) inhibitors on the ktr in ultra-thin non-failing and failing human right ventricular trabeculae. After stabilizing the muscle either at L90 (90% of optimal length) or at Lopt (optimal length), we rapidly changed the length to either Lopt or L90 and measured ktr. We report that length-dependent changes in ktr occur very rapidly (in the order of seconds or faster) in both non-failing and failing muscles and that the length at which a muscle had been stabilized prior to the length change does not significantly affect ktr. In addition, at L90 and at Lopt, PKA and PKC-βII inhibitors did not significantly change ktr. Our results reveal that length-dependent regulation of cross-bridge cycling kinetics predominantly occurs rapidly and involves the intrinsic properties of the myofilament rather than post-translational modifications that are known to occur in the cardiac muscle as a result of a change in muscle/sarcomere length.

Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

Learning and memory: conditions for fear.

State-dependent fear conditioning can be induced by activation of hippocampal extrasynaptic GABAA receptors and is regulated by protein kinase CβII activity and microRNA-33 signalling.

GABAergic mechanisms regulated by miR-33 encode state-dependent fear.

Fear-inducing memories can be state dependent, meaning that they can best be retrieved if the brain states at encoding and retrieval are similar. Restricted access to such memories can present a risk for psychiatric disorders and hamper their treatment. To better understand the mechanisms underlying state-dependent fear, we used a mouse model of contextual fear conditioning. We found that heightened activity of hippocampal extrasynaptic GABAA receptors, believed to impair fear and memory, actually enabled their state-dependent encoding and retrieval. This effect required protein kinase C-βII and was influenced by miR-33, a microRNA that regulates several GABA-related proteins. In the extended hippocampal circuit, extrasynaptic GABAA receptors promoted subcortical, but impaired cortical, activation during memory encoding of context fear. Moreover, suppression of retrosplenial cortical activity, which normally impairs retrieval, had an enhancing effect on the retrieval of state-dependent fear. These mechanisms can serve as treatment targets for managing access to state-dependent memories of stressful experiences.

Circulating microparticles from patients with valvular heart disease and cardiac surgery inhibit endothelium-dependent vasodilation

ObjectiveVascular function is very important for maintaining circulation after cardiac surgery. Circulating microparticles (MPs) generated in various diseases play important roles in causing inflammation, coagulation, and vascular injury. However, the impact of MPs generated from patients who have valvular heart disease (VHD), before and after cardiac surgery, on vascular function remains unknown. This study is designed to investigate the impact of such MPs on vasodilation. MethodsMicroparticles were isolated from age-matched healthy subjects and patients who had VHD, before cardiac surgery, and at 12 hours and 72 hours afterward. The number of MPs was measured and compared. Effects evaluated were of the impact of MPs on: vasodilation of mice aorta; the phosphorylation and expression of Akt, endothelial nitric oxide synthase (eNOS), protein kinase C-βII (PKC-βII), and p70 ribosomal protein S6 kinase (p70S6K); expression of caveolin-1; the association of eNOS with heat shock protein 90 (HSP90); and generation of nitric oxide and superoxide anion of human umbilical vein endothelial cells. ResultsCompared with the healthy subjects, VHD patients had significantly higher levels of circulating MPs and those MPs before cardiac surgery can: impair endothelium-dependent vasodilation; inhibit phosphorylation of Akt and eNOS; increase activation of PKC-βII and p70S6K; enhance expression of caveolin-1; reduce the association of HSP90 with eNOS; decrease nitric oxide production, and increase superoxide anion generation. These deleterious effects were even stronger in postoperative MPs. ConclusionsOur data demonstrate that MPs generated from VHD patients before and after cardiac surgery contributed to endothelial dysfunction, by uncoupling and inhibiting eNOS. Circulating MPs are potential therapeutic targets for the maintenance of vascular function postoperatively.

Correlation of Lower Concentrations of Hydrogen Sulfide with Activation of Protein Kinase CβII in Uremic Accelerated Atherosclerosis Patients.

Background:Hydrogen sulfide (H2S) plays a protective role in chronic hemodialysis (CHD) patients. In this study, we further investigate the relationship between H2S and conventional protein kinase CβII (cPKCβII) in CHD patients with uremic accelerated atherosclerosis (UAAS).Methods:A total of 30 healthy people, 30 CHD patients without AS and 30 CHD patients with AS (CHD + AS) were studied. Plasma H2S was measured with a sulfide sensitive electrode, and cPKCβII membrane translocation was detected by Western blotting.Results:Plasma H2S in CHD + AS group was significantly lower than that in CHD patients. cPKCβII membrane translocation in CHD + AS group increased significantly compared with CHD group. Plasma H2S concentration was negatively correlated with cPKCβII membrane translocation in CHD + AS patients.Conclusions:These findings suggest a possible linkage between H2S metabolism and cPKCβII activation, which may contribute to the development of UAAS in CHD patients.