Complex Regulation of PKCβ2 and PDK-1/AKT by ROCK2 in Diabetic Heart

Objectives

The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKCβ2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKCβ2, and to determine if their interaction affects PDK-1/Akt signaling.

Methods

Regulation by ROCK of PKCβ2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKCβ2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKCβ2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy.

Results

Expression of ROCK2 and expression and phosphorylation of PKCβ2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal immunoprecipitation, while ROCK2 directly phosphorylated PKCβ2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKCβ2 T641 phosphorylation, and this was associated with attenuation of PKCβ2 activity. PKCβ2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts.

Conclusion

This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKCβ2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes.     

Down-regulation of PKCζ in renal cell carcinoma and its clinicopathological implications

Background

Metastatic renal cell carcinoma (RCC) is highly resistant to systemic chemotherapy. Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC. Recent studies have shown the atypical PKCζ is an important regulator of tumorigenesis. However, the correlation between PKCζ expression and the clinical outcome in RCC patients is unclear. We examined the level of PKCζ expression in human RCC.

Methods

PKCζ mRNA and protein expressions were examined by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) respectively in RCC tissues of 144 patients. Cellular cytotoxicity and proliferation were assessed by MTT.

Results

PKCζ expression was significantly higher in normal than in cancerous tissues (P < 0.0001) by real-time PCR and IHC. Similarly, PKCζ expression was down-regulated in four renal cancer cell lines compared to immortalized benign renal tubular cells. Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P = 0.04), but no such association was found in TNM stage (P = 0.13). Tumors with higher PKCζ expression were associated with tumor size (P = 0.048). Expression of higher PKCζ found a poor survival in patients with high tumor grade. Down-regulation of PKCζ showed the significant chemoresistance in RCC cell lines. Inactivation of PKCζ expression enhanced cellular resistance to cisplatin and paclitaxel, and proliferation in HK-2 cells by specific PKCζ siRNA and inhibitor.

Conclusions

PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.     

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy

Aims/hypothesis

Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia.

Methods

Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor.

Results

PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death.

Conclusions

In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.     

Differential Roles of the PKC Novel Isoforms,PKCδ and PKCε, in Mouse and Human Platelets

Background

Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

Methodology/Principal Findings

In this study, we focus on the role of two novel PKC isoforms, PKCδ and PKCε, in both mouse and human platelets. PKCδ is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCδ undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCε, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCε in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRγ-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor.

Conclusions

These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms δ and ε in human and mouse platelets and a selective role for PKCε in signalling through GPVI.     

Protein Kinase C ε Expression in Platelets from Patients with Acute Myocardial Infarction

Objective

Platelets play crucial roles in the pathophysiology of thrombosis and myocardial infarction. Protein kinase C ε (PKCε) is virtually absent in human platelets and its expression is precisely regulated during human megakaryocytic differentiation. On the basis of what is known on the role of platelet PKCε in other species, we hypothesized that platelets from myocardial infarction patients might ectopically express PKCε with a pathophysiological role in the disease.

Methods and Results

We therefore studied platelet PKCε expression from 24 patients with myocardial infarction, 24 patients with stable coronary artery disease and 24 healthy subjects. Indeed, platelets from myocardial infarction patients expressed PKCε with a significant frequency as compared to both stable coronary artery disease and healthy subjects. PKCε returned negative during patient follow-up. The forced expression of PKCε in normal donor platelets significantly increased their response to adenosine diphosphate-induced activation and adhesion to subendothelial collagen.

Conclusions

Our data suggest that platelet generations produced before the acute event retain PKCε-mRNA that is not down-regulated during terminal megakaryocyte differentiation. Results are discussed in the perspective of peri-infarctual megakaryocytopoiesis as a critical component of myocardial infarction pathophysiology.     

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils

Background

PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils.

Results

We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd^−/− mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice.

Conclusions

These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.     

Clinicopathological Correlation and Prognostic Significance of Protein Kinase Cα Overexpression in Human Gastric Carcinoma

Objectives

This study investigated the PKCα protein expression in gastric carcinoma, and correlated it with clinicopathological parameters. The prognostic significance of PKCα protein expression in gastric carcinoma was analyzed.

Methods

Quantitative real-time PCR test was applied to compare the PKCα mRNA expression in tumorous and nontumorous tissues of gastric carcinoma in ten randomly selected cases. Then PKCα protein expression was evaluated in 215 cases of gastric carcinoma using immunohistochemical method. The immunoreactivity was scored semiquantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely PKCα overexpression group with score 2 or 3, and non-overexpression group with score 0 or 1. The PKCα protein expression was correlated with clinicopathological parameters. Survival analysis was performed to determine the prognostic significance of PKCα protein expression in patients with gastric carcinoma.

Results

PKCα mRNA expression was upregulated in all ten cases of gastric carcinoma via quantitative real-time PCR test. In immunohistochemical study, eighty-eight out of 215 cases (41%) of gastric carcinoma revealed PKCα protein overexpression, which was statistically correlated with age (P = 0.0073), histologic type (P<0.0001), tumor differentiation (P = 0.0110), depth of invasion (P = 0.0003), angiolymphatic invasion (P = 0.0373), pathologic stage (P = 0.0047), and distant metastasis (P = 0.0048). We found no significant difference in overall and disease free survival rates between PKCα overexpression and non-overexpression groups (P = 0.0680 and 0.0587). However, PKCα protein overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 0.632, P = 0.0415).

Conclusions

PKCα protein is upregulated in gastric carcinoma. PKCα protein expression is statistically correlated with age, histologic type, tumor differentiation, depth of invasion, angiolymphatic invasion, pathologic stage, and distant metastasis. The PKCα protein overexpression in patients with gastric carcinoma is a significant independent prognostic factor in multivariate Cox regression analysis.     

The CCL2/CCR2 Axis Enhances Vascular Cell Adhesion Molecule-1 Expression in Human Synovial Fibroblasts

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of osteoarthritis (OA). Here, we investigated the intracellular signaling pathways involved in CCL2-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human OA synovial fibroblasts (OASFs).

Methodology/Principal Findings

Stimulation of OASFs with CCL2 induced VCAM-1 expression. CCL2-mediated VCAM-1 expression was attenuated by CCR2 inhibitor (RS102895), PKCδ inhibitor (rottlerin), p38MAPK inhibitor (SB203580), and AP-1 inhibitors (curcumin and tanshinone IIA). Stimulation of cells with CCL2 increased PKCδ and p38MAPK activation. Treatment of OASFs with CCL2 also increased the c-Jun phosphorylation and c-Jun binding to the AP-1 element on the VCAM-1 promoter. Moreover, CCL2-mediated CCR2, PKCδ, p38MAPK, and AP-1 pathway promoted the adhesion of monocytes to the OASFs monolayer.

Conclusions/Significance

Our results suggest that CCL2 increases VCAM-1 expression in human OASFs via the CCR2, PKCδ, p38MAPK, c-Jun, and AP-1 signaling pathway. The CCL2-induced VCAM-1 expression promoted monocytes adhesion to human OASFs.     

Serum TGF-β1 as a Biomarker for Type 2 Diabetic Nephropathy: A Meta-Analysis of Randomized Controlled Trials

Background

Abnormal expression of serum TGF-β1 was found in patients with diabetic nephropathy. However, the association of TGF-β1 with the risk of diabetic nephropathy remains unknown. The present study was undertaken to investigate whether such an association exists.

Methods

We searched the Chinese VIP, Wangfang, China National Knowledge Infrastructure, PubMed, Embase, and Google Scholar databases for relevant studies and extracted all eligible data. Stata12 software was used for statistical analysis.

Results

Nine reports met our criteria and were used for data extraction. There were 264 patients and 227 healthy controls from qualified reports in this meta-analysis. The results suggested that serum TGF-β1 levels were significantly up-regulated in patients with diabetic nephropathy; the instrumental variable was 3.94 (95% confidence interval 3.20–4.68, p<0.01).

Conclusions

Meta-analysis suggested that elevated serum TGF-β level in patients with diabetes is associated with a high risk of nephropathy. Further studies are required to validate these observations.     

Involvement of the Tubular ClC-Type Exchanger ClC-5 in Glomeruli of Human Proteinuric Nephropathies

Glomerular protein handling mechanisms have received much attention in studies of nephrotic syndrome. Histopathological findings in renal biopsies from severely proteinuric patients support the likelihood of protein endocytosis by podocytes. ClC-5 is involved in the endocytosis of albumin in the proximal tubule.

Aim

To investigate whether ClC-5 is expressed in the glomerular compartment and whether it has a role in proteinuric nephropathies. ClC-5 expression was studied using Real-time PCR in manually- and laser-microdissected biopsies from patients with type 2 diabetes (n 37) and IgA nephropathy (n 10); in biopsies of membranous glomerulopathy (MG) (n 14) immunohistochemistry for ClC-5 (with morphometric analysis) and for WT1 was done. Controls: cortical tissue (n 23) obtained from unaffected parts of tumor-related nephrectomy specimens.

Results

ClC-5 was expressed at glomerular level in all biopsies. Glomerular ClC-5 levels were significantly higher in diabetic nephropaty and MG at both mRNA and protein level (p<0.002; p<0.01). ClC-5 and WT1 double-staining analysis in MG showed that ClC-5 was localized in the podocytes. ClC-5 ultrastructural immunolocalization was demonstrated in podocytes foot processes. Our study is the first to demonstrate that ClC-5 is expressed in human podocytes. The ClC-5 overexpression found in biopsies of proteinuric patients suggests that proteinuria may play a part in its expression and that podocytes are likely to have a key role in albumin handling in proteinuric states.     

An update: the role of Nephrin inside and outside the kidney

Nephrin is a key molecule in podocytes to maintain normal slit diaphragm structure. Nephin interacts with many other podocyte and slit diaphragm protein and also mediates important cell signaling pathways in podocytes. Loss of nephrin during the development leads to the congenital nephrotic syndrome in children. Reduction of nephrin expression is often observed in adult kidney diseases including diabetic nephropathy and HIV-associated nephropathy. The critical role of nephrin has been confirmed by different animal models with nephrin knockout and knockdown. Recent studies demonstrate that knockdown of nephrin expression in adult mice aggravates the progression of unilateral nephrectomy and Adriamycin-induced kidney disease. In addition to its critical role in maintaining normal glomerular filtration unit in the kidney, nephrin is also expressed in other organs. However, the exact role of nephrin in kidney and extra-renal organs has not been well characterized. Future studies are required to determine whether nephrin could be developed as a drug target to treat patients with kidney disease.     

Glycoxidised LDL Induced the Upregulation of Axl Receptor Tyrosine Kinase and Its Ligand in Mouse Mesangial Cells

Aim/Hypothesis

Low-density lipoprotein (LDL) is subjected to glycoxidation in diabetes, and a novel signalling mechanism by which glycoxidised LDL functions in glomerular mesangial cells remains to be ascertained.

Methods

We performed gene expression analysis in mouse glomerular mesangial cells treated with LDL modified by glycation and oxidation (GO-LDL, 100 µg/ml) for 48 h by using DNA microarray analysis and quantitative real-time PCR. We examined the GO-LDL-specific changes in gene and protein expression in mesangial cells and glomeruli of type 2 diabetic Zucker diabetic fatty (ZDF) rats.

Results

By microarray profiling, we noted that GO-LDL treatment increased Axl receptor tyrosine kinase (Axl) mRNA expression (∼2.5-fold, p<0.05) compared with normal LDL (N-LDL) treatment in mesangial cells. Treatment with GO-LDL also increased the protein levels of Axl and its ligand Gas6 as measured by Western blotting. These increases were inhibited by neutralising Axl receptor-specific antibody. Silencing Gas6 by siRNA inhibited GO-LDL-induced Axl expression in mesangial cells. Axl and Gas6 protein were also increased in cells cultured in high glucose (30 mM) or methylglyoxal (200 µM). Gas6 treatment increased the expression and secretion of TGF-β1 protein, a key regulator of extracellular matrix expression in the glomeruli of diabetic kidneys. Immunohistochemical analyses of glomeruli from 20-week-old ZDF rats exhibited increased Axl protein expression. Rottlerin, a selective PKC-δ inhibitor, completely blocked Gas6-induced TGF-β1 expression.

Conclusions/Interpretation

These data suggest that LDL modified by glycoxidation may mediate Axl/Gas6 pathway activation, and this mechanism may play a significant role in the pathogenesis of diabetic nephropathy.     

IL-33 Attenuates Anoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis by Inhibition of PKCβ/JNK Pathway

Background

Interleukin-33 (IL-33) is a new member of the IL-1 cytokine family. The objectives of present study are to assess whether IL-33 can protect cardiomyocytes from anoxia/reoxygenation (A/R)-induced injury and the mechanism involved in the protection.

Methods

Cardiomyocytes derived from either wild type or JNK1^−/− mice were challenged with an A/R with or without IL-33. Myocyte apoptosis was assessed by measuring caspase 3 activity, fragmented DNA and TUNEL staining. In addition, cardiomyocyte oxidative stress was assessed by measuring DHR123 oxidation; PKCβII and JNK phosphorylation were assessed with Western blot.

Results

Challenge of cardiomyocytes with an A/R resulted in cardiomyocyte oxidative stress, PKCβII and JNK phosphorylation, and myocyte apoptosis. Treatment of the cardiomyocytes with IL-33 attenuated the A/R-induced myocyte oxidative stress, prevented PKCβII and JNK phosphorylation and attenuated the A/R-induced myocyte apoptosis. The protective effect of the IL-33 did not show in cardiac myocytes with siRNA specific to PKCβII or myocytes deficient in JNK1. Inhibition of PKCβII prevented the A/R-induced JNK phosphorylation, but inhibition of JNK1 showed no effect on A/R-induced PKCβII phosphorylation.

Conclusions

Our results indicate that IL-33 prevents the A/R-induced myocyte apoptosis through inhibition of PKCβ/JNK pathway.     

Ligation of α-Dystroglycan on Podocytes Induces Intracellular Signaling: A New Mechanism for Podocyte Effacement?

Background

α-Dystroglycan is a negatively charged glycoprotein that covers the apical and basolateral membrane of the podocyte. Its transmembrane binding to the cytoskeleton is regulated via tyrosine phosphorylation (pY892) of β-dystroglycan. At the basolateral side α-dystroglycan binds the glomerular basement membrane. At the apical membrane, it plays a role in the maintenance of the filtration slit. In this study, we evaluated whether ligation of α-dystroglycan with specific antibodies or natural ligands induces intracellular signaling, and whether there is an effect on podocyte architecture.

Methodology/Principal Findings

Conditionally immortalized podocytes were exposed in vitro to antibodies to α-dystroglycan, and to fibronectin, biglycan, laminin and agrin. Intracellular calcium fluxes, phosphorylation of β-dystroglycan and podocyte architecture were studied. Antibodies to α-dystroglycan could specifically induce calcium signaling. Fibronectin also induced calcium signaling, and led to dephosphorylation of pY892 in β-dystroglycan. Ligation of α-dystroglycan resulted in an altered actin architecture, a decreased number of podocyte pedicles and a more flattened appearance of the podocyte.

Conclusions/Significance

We conclude that ligation of α-dystroglycan on podocytes induces intracellular calcium signaling, which leads to an altered cytoskeleton architecture akin to the situation of foot process effacement. In particular the ability of fibronectin to induce intracellular signaling events is of interest, since the expression and excretion of this protein is upregulated in several proteinuric diseases. Therefore, fibronectin-induced signaling via dystroglycan may be a novel mechanism for foot process effacement in proteinuric diseases.     

Sulodexide Decreases Albuminuria and Regulates Matrix Protein Accumulation in C57BL/6 Mice with Streptozotocin-Induced Type I Diabetic Nephropathy

Objective

Sulodexide is a mixture of glycosaminoglycans that may reduce proteinuria in diabetic nephropathy (DN), but its mechanism of action and effect on renal histology is not known. We investigated the effect of sulodexide on disease manifestations in a murine model of type I DN.

Methods

Male C57BL/6 mice were rendered diabetic with streptozotocin. After the onset of proteinuria, mice were randomized to receive sulodexide (1 mg/kg/day) or saline for up to 12 weeks and renal function, histology and fibrosis were examined. The effect of sulodexide on fibrogenesis in murine mesangial cells (MMC) was also investigated.

Results

Mice with DN showed progressive albuminuria and renal deterioration over time, accompanied by mesangial expansion, PKC and ERK activation, increased renal expression of TGF-β1, fibronectin and collagen type I, III and IV, but decreased glomerular perlecan expression. Sulodexide treatment significantly reduced albuminuria, improved renal function, increased glomerular perlecan expression and reduced collagen type I and IV expression and ERK activation. Intra-glomerular PKC-α activation was not affected by sulodexide treatment whereas glomerular expression of fibronectin and collagen type III was increased. MMC stimulated with 30 mM D-glucose showed increased PKC and ERK mediated fibronectin and collagen type III synthesis. Sulodexide alone significantly increased fibronectin and collagen type III synthesis in a dose-dependent manner in MMC and this increase was further enhanced in the presence of 30 mM D-glucose. Sulodexide showed a dose-dependent inhibition of 30 mM D-glucose-induced PKC-βII and ERK phosphorylation, but had no effect on PKC-α or PKC-βI phosphorylation.

Conclusions

Our data demonstrated that while sulodexide treatment reduced proteinuria and improved renal function, it had differential effects on signaling pathways and matrix protein synthesis in the kidney of C57BL/6 mice with DN.     

PKCδ Localization at the Membrane Increases Matrix Traction Force Dependent on PLCγ1/EGFR Signaling

Introduction

During wound healing, fibroblasts initially migrate into the wound bed and later contract the matrix. Relevant mediators of transcellular contractility revealed by systems analyses are protein kinase c delta/myosin light chain-2 (PKCδ/MLC-2). PKCδ is activated by growth factor-driven PLCγ1 hydrolysis of phosphoinositide bisphosphate (PIP₂) hydrolysis when it becomes tranlocated to the membrane. This leads to MLC-2 phosphorylation that regulates myosin for contractility. Furthermore, PKCδ n-terminus mediates PKCδ localization to the membrane in relative proximity to PLCγ1 activity. However, the role this localization and the relationship to its activation and signaling of force is not well understood. Therefore, we investigated whether the membrane localization of PKCδ mediates the transcellular contractility of fibroblasts.

Methods

To determine PKCδ activation in targeted membrane locations in mouse fibroblast cells (NR6-WT), two PKCδ constructs were generated; PKCδ-CaaX with farnesylation moiety targeting PKCδ to the membrane and PKCδ-SaaX a non-targeting control.

Results

Increased mean cell force was observed before and during EGF stimulation in fibroblasts expressing membrane-targeted PKCδ (PKCδ-CaaX) when analyzed with 2D cell traction force and 3D compaction of collagen matrix. This effect was reduced in cells deficient in EGFR/PLCy1 signaling. In cells expressing non-membrane targeted PKCδ (PKCδ-SaaX), the cell force exerted outside the ECM (extracellular matrix) was less, but cell motility/speed/persistence was increased after EGF stimulation. Change in cell motility and increased force exertion was also preceded by change in cell morphology. Organization of actin stress fibers was also decreased as a result of increasing membrane targeting of PKCδ.

Conclusion

From these results membrane tethering of PKCδ leads to increased force exertion on ECM. Furthermore, our data show PLCγ1 regulation of PKCδ, at least in part, drives transcellular contractility in fibroblasts.     

Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase Cε-Dependent Mechanism in Neurons

Background

The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction.

Methods

In primary cortical cell cultures from newborn rats’ brains, live cell light microscopy was used to measure neurite retraction after propofol (2 µM) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKCε translocation inhibitor peptide. Changes in PKCε Ser⁷²⁹ phosphorylation were detected with Western blot.

Results

The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKCε translocation inhibitor peptide. OA increases via PLD and propofol decreases PKCε Ser⁷²⁹ phosphorylation, a crucial step in the activation of PKCε.

Conclusions

Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKCε-mediated pathway, and PKCε maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge.