PKC-ε Research Articles

Novel role of PKC epsilon in mitotic spindle stability

Mitosis is a highly regulated process characterized by dramatic and coordinated morphological changes to ensure the fidelity of chromosome segregation. Missegregation of mitotic chromosomes leads to a condition that underlies chromosomal instability(1), which is a hallmark of cancer. In order to assure symmetry and bipolarity of the cell division process, mitotic spindle microtubules properly segregate mitotic chromosomes (2). Among the several isoforms of serine/threonine kinases, PKCe is one of the best understood for its role as a transforming oncogene, and it has been found overexpressed in different types of tumors. In 2008, Saurin and colleagues demonstrated the involvement of PKCe in the regulation of the late stage of mitosis (3). Through its association with 14-3-3 at the midbody, PKCe is essential for the successful completion of cytokinesis, and the inhibition of functional PKCe-14-3- 3 complex leads to abscission failure and multinucleated phenotype in cells. In this study, we found that PKCe is involved in mitotic spindle stability. Using fluorescence microscopy, we found that the active form of PKCe (phosphorylated at Ser-729), colocalizes to the centrosome in cells in metaphase, where the mitotic spindle nucleation occurs. Furthermore, experiments of co-immunoprecipitation revealed that, when cells are synchronized in metaphase, PKCɛ is associated to ɣ-tubulin, a member of the tubulin superfamily localized to the microtubule organizing centers and is essential for microtubule nucleation from centrosomes. Consequently modulation of PKCɛ expression affects spindle stability: PKCɛ downregulation by specific shRNA results in mitotic spindle disorganization with a reduction of the amount of centrosomal and mitotic ɣ-Tubulin and αβ-tubulin fluorescence. Mitotic spindle formation assays using Nocodazole, known to interfere with the polymerization of microtubules, revealed that cells lacking PKCɛ were unable to regrow microtubules after depolymerization. These results reveal a novel role of PKCɛ in mitotic spindle stability, which likely determinant for genome stability.

Novel role of PKC ɛ in mitotic spindle stability

Mitosis is a highly regulated process characterized by dramatic and coordinated morphological changes to ensure the fidelity of chromosome segregation. Misseg- regation of mitotic chromosomes leads to a condition that underlies chromosomal instability(1), which is a hallmark of cancer. In order to assure symmetry and bipo- larity of the cell division process, mitotic spindle microtubules properly segregate mitotic chromosomes (2). Among the several isoforms of serine/threonine kinases, PKCe is one of the best understood for its role as a transforming oncogene, and it has been found overexpressed in different types of tumors. In 2008, Saurin and col- leagues demonstrated the involvement of PKCe in the regulation of the late stage of mitosis (3). Through its association with 14-3-3 at the midbody, PKCe is essential for the successful completion of cytokinesis, and the inhibition of functional PKCe-14-3- 3 complex leads to abscission failure and multinucleated phenotype in cells. In this study, we found that PKCe is involved in mitotic spindle stability. Using fluorescence microscopy, we found that the active form of PKCe (phosphorylated at Ser-729), co- localizes to the centrosome in cells in metaphase, where the mitotic spindle nuclea- tion occurs. Furthermore, experiments of co-immunoprecipitation revealed that, when cells are synchronized in metaphase, PKCɛ is associated to ɣ-tubulin, a member of the tubulin superfamily localized to the microtubule organizing centers and is essen- tial for microtubule nucleation from centrosomes. Consequently modulation of PKCɛ expression affects spindle stability: PKCɛ downregulation by specific shRNA results in mitotic spindle disorganization with a reduction of the amount of centrosomal and mitotic ɣ-Tubulin and αβ-tubulin fluorescence. Mitotic spindle formation assays using Nocodazole, known to interfere with the polymerization of microtubules, revealed that cells lacking PKCɛ were unable to regrow microtubules after depolymerization. These results reveal a novel role of PKCɛ in mitotic spindle stability, which likely determinant for genome stability.

PKCε regulates vessel formation by peri-vascular adipose tissue (PVAT) cells

Vessel formation is crucial in tumour growth and tissue regeneration. Protein kinase C (PKC) e has a well-known role on hematopoietic and mesenchymal progenitor cell differentiation and proliferation (Gobbi et al. 2013). Although PKCe has a demonstrated role in vascular restenosis, data on PKCe and vascular progenitor differentiation are still lacking. The aim of this work was to study the role of PKCe in vessel formation by adult adipose tissue cell progenitors. We, first, isolated the vessel progenitors from the adipose tissue localized between aortic arch and pulmonary artery of adult mice by collagenase/elastase digestion followed by magnetic immunoselection of Sca1+ cells (Passmann et al. 2008). We, then, tested their capability to form vessels in collagen gels and to differentiate to endothelial and smooth muscle lineage after treatment with PKCe specific activator and inhibitor peptides. The functional experiments showed that the pharmacological activation of endogenous PKCe abrogated tubule formation with a concomitant decrease of smooth alpha-actin (SMA) and platelet endothelial cell adhesion molecule (PECAM) together with the up-regulation of p-PAK1 expression. In vivo transient over-expression of PKCe significantly reduced SMA and PECAM expression levels in vessel wall cells. Together our data suggests that PKCe may affect vessel wall remodelling balancing the “phenotypic switching” (Salmon et al. 2013) between the proliferative and the differentiated state of smooth muscle and endothelial progenitor mesenchymal cells.

Correction: Calreticulin Induces Dilated Cardiomyopathy

In figure 1A □MHC should be replaced with αMHC in all 6 places. Please see the corrected figure 1 here: In figure 1B □MHC should be replaced with αMHC in 2 places. Please see the corrected figure 1 here: In figure 2A □MHC should be replaced with αMHC in 2 places. Please see the corrected figure 2 here: In figure 6B PKC□ should be replaced with PKCe in 1 place. Please see the corrected figure 6 here: In figure 7C □-gal should be replaced with β-gal in 1 place. Please see the corrected figure 7 here:

Role of caveolin‐3 in adenosine‐induced increase in mitochondrial PKCε

Activation of adenosine receptors has been shown to promote translocation of PKC isoforms to both caveolar plasma membrane and mitochondria. Adenosine receptors, PKC and caveolin‐3 play important roles in the cardioprotection of ischemic preconditioning. The present study was designed to determine whether adenosine receptor activation‐induced translocation of PKCε to mitochondria is caveolin‐3‐dependent. Immunofluorescence imaging of isolated mitochondria showed that adenosine‐induced increase in mitochondria PKCε was inhibited by adenosine A1 receptor specific inhibitor DPCPX (36.0 ± 1.2% vs. 72.7 ± 1.8% in cardiomyocytes and 19.0 ± 1.0% vs 57.5 ± 2.0% in H9c2 cells, n=3, P<0.01). Neither adenosine A2B receptor specific inhibitor MRS1754 nor PI3K kinase inhibitor Wortmannin prevented adenosine‐induced increase in mitochondrial PKCε. Interestingly, adenosine‐induced mitochondrial translocation of PKCε was significantly reduced by suppressing caveolin‐3 expression with specific siRNA (52.0 ± 2.0% vs. 17.7 ± 2.0%, adenosine vs. adenosine/caveolin‐3‐siRNA in H9c2 cells, n=3, P<0.01), while negative siRNA had no effect. In conclusion, we demonstrate that adenosine receptor activation induces caveolin‐3‐dependent translocation of PKCε to mitochondria. These results point out a novel mechanism in regulating PKC isoform in mitochondria.

Activation and nuclear translocation of PKCε promotes skeletal muscle cell differentiation via HMGA1 downregulation

The role of novel PKCs in skeletal muscle differentiation has recently emerged. PKCθ is the most expressed isoform of PKCs in muscle and it promotes the fusion of myoblasts [1]. Recently, we have demonstrated that PKCe is implicated in myocardiocyte differentiation of bone marrow mesenchymal stem cells [2] but the role of PKCe in skeletal muscle cell regeneration has only recently emerged [3]. We here demonstrate that both nuclear and cytoplasmic fractions of PKCe are up-regulated during in vitro C2C12 cell line and satellite cell differentiation. We also show that PKCe is able to modulate myogenic differentiation genes via a downmodulation of HMGA1 proteins that promotes myogenin accumulation and mature myoblast formation. To study the effects of PKCe on muscle regeneration, we have used the in vivo model of CTX-induced skeletal muscle injury. We show that the upregulation of PKCe also occurs in vivo, particularly in the centro-nucleated regenerating fibers that are derived from the fusion process of the resident satellite cells, suggesting a role for PKCe in human satellite cell-driven muscle repair and substitution, with clinically relevant implications in human muscle pathology.

Protein kinase C (PKC)ε and human CD4 T cell proliferation

T-lymphocytes contain up to eight different PKC isotypes and PKCθ has become the most interesting isotype for T-cell activation, proliferation, and transforming growth factor (TGF)-1β signalling [1]. However, It has been suggested that also PKCe may have a role in inflammation and immune-mediated disorders [2]. Thus, we have analyzed the ability of PKCe to control human CD4+ T cell proliferation and their sensitivity to TGF-1β. We demonstrate a nonredundant role of PKCe in CD4+ T cell proliferation triggered in vitro by CD3 stimulation. PKCe sustains NF-kB and, consequently, IL-2 receptor chains transcription and CD25 cell surface expression levels. Moreover, PKCe silencing potentiates the inhibitory effects of TGF-1β, affecting Smad2 phosphorylation levels. Finally, assuming that PKCe could be involved in CD4+ T cell mediated-autoimmune diseases, we have isolated CD4 T cells from Hashimoto Thyroiditis (HT) patients an autoimmune disorder characterized by reduced serum concentration of TGF-1β and TReg cell subsets with defective suppressive functions [3,4]. In HT CD4+ T cells we found a significant increase of PKCe expression, accounting for their decreased sensitivity to TGF-1β. The potentially new roles of PKCe in the pathophysiology of HT and Th/Treg polarization are discussed.

Effect of calcium-sensing receptor in cardiac hypertrophy induced by angiotensin II in cultured neonatal rat cardiomyocytes

Objective To explore the roles and possible mechanism of calcium-sensing receptor(CaSR) in cell cardiac hypertrophy model using angiotensin Ⅱ (Ang Ⅱ ).Methods The cultured neonatal rat ventricular myocytes were treated with Ang Ⅱ as cell cardiac hypertrophy model.Hypertrophic neonatal rat cardiomyocytes were treated with GdCl3(a specific agonist of CaSR) and/or with Ro318220(a specific inhibitor of PKC pathway).To evaluate the status of cardiac hypertrophy,cell diameter was observed by HE dyeing,and protein content was determined through coomassie brilliant blue protein kit.The intracellular calcium concentration( [ Ca2+]i) was determined by laser scanning confocal microscope.The protein expression of CaSR and PKC pathway were analyzed using Western blotting.Results ①Compared to the control group(0.1263 ± 0.0443),the protein expression of CaSR was increased in Ang Ⅱ group and in GdCl3 group(0.1963 ± 0.0375,0.2778 ± 0.0564,all P＜ 0.05).Moreover,compared with Ang Ⅱ alone,the increase was significant in GdCl3 group(P ＜ 0.05).②Compared to control group(222.70 ± 22.09),AngⅡ group(392.16 ± 36.85) remarkably increased [Ca2+]i(P＜ 0.05),and this increase of [Ca2+]i was further enhanced in GdCl3 group (502.60 ± 44.21) versus Ang Ⅱ group (P ＜ 0.05).③Compared to control group,Ang Ⅱ could induce cardiomyocyte hypertrophy,and GdCl3 enhanced the effect.Moreover,this enhancement was attenuated by Ro318220.④Compared to control group(0.27 ± 0.07,0.69 ± 0.06,0.87 ± 0.04),the protein expression of PKCα,PKCe and PKCδ was increased in Ang Ⅱ group(0.60 ± 0.16,1.02 ± 0.13,1.20 ± 0.18,all P＜ 0.05) and the protein expression of PKCα,PKCe was increased in GdCl3 group(0.82 ± 0.16,1.34 ± 0.12,all P ＜ 0.05).Moreover,compared with Ang Ⅱ group,the protein expression of PKCα,PKCe was obviously increased in GdCl3 group (all P ＜ 0.05);compared with GdCl3 group,the protein expression of PKCα,PKCe(0.41 ± 0.10,0.85 ± 0.14) was obviously decreased in Ro318220 group(all P ＜ 0.05).Conclusions CaSR is involved in cardiac hypertrophy induced by Ang Ⅱ through PKC pathway in cultured neonatal rat cardiomyocytes. Key words: Receptors, calcium-sensing; Protein kinase C; Angiotensin Ⅱ ; Cardiac hypertrophy

Role of protein kinase C in induction of vascular endothelial growth factor secretion by isoflurane in primary cultured rat cardiomyocytes

Objective To investigate the role of protein kinase C (PKC) in induction of vascular endothelial growth factor (VEGF) secretion by isoflurane in primary cultured rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 6 groups ( n = 6 each): control group (group C), 3 different concentration isoflurane groups (group Ⅰ1-3 ), PKC inhibitor calphostin C group (group P), and PKC inhibitor + isoflurane group (group PI). The cells were exposed to 0.7%, 1.4% and 2.1% isoflurane for6 h in group Ⅰ1-3 respectivly. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L in group P. Calphostin C was added to the culture medium with a final concentration of 50 nmol/L, then the cells were exposed to 1.4% isoflurane for 6 h in group PI. VEGF concentrations and expression of PKC isoforms were determined by ELISA and Western blot respectively. Results Compared with group C, the VEGF concentration was significantly increased in group Ⅰ2 and Ⅰ3, and PKCe expression was down-regulated in the cytoplasm while upregulated in the cytomembrane in group Ⅰ2 ( P ＜ 0.01 ), but no significant change was found in the parameters mentioned above in group Ⅰ2 ( P ＞ 0.05). PKCα, PKCδ and PKCζ expression was significantly higher in the cytoplasm than in the cytomembrane in group C and Ⅰ2. VEGF concentrations were gradually increased with the increase in isoflurane concentrations ( P ＜ 0.05). VEGF concentrations were significantly lower in group PI than in Ⅰ2 ( P ＜0.05) .Conclusion Isoflurane induces VEGF secretion in primary cultured rat cardiomyocytes through translocation of PKCe from the cytoplasm to the cytomembrane, suggesting that it is a mechanism of the cardioprotective effects of isoflurane. Key words: Isoflurane; Myocytes, cardiac; Protein kinase C; Vascular endothelial growth factors

Specific involvement of PKC-ε in the synergistic interaction between agonists acting at alpha-2-adrenergic and delta-opioid receptors in spinal cord

Specific involvement of PKC-ε in the synergistic interaction between agonists acting at alpha-2-adrenergic and delta-opioid receptors in spinal cord

Role of mitochondrial ATP-sensitive potassium channel in sevoflurane preconditioning-reduced focal cerebral ischemia-reperfusion injury in rats

Objective To investigate the role of the mitochondrial ATP-sensitive potassium (mito-K_(ATP)) channel in sevoflurane preconditioning-reduced focal cerebral ischemia-reperfnsion (I/R) injury in rats. Methods One hundred healthy 3-4 month old male SD rats 250-300 g were randomly assigned into 5 groups (n = 20 each) : group Ⅰ sham operatiun (group S); group Ⅱ I/R; group Ⅲ sevoflurane preconditioning (group Sevo); group Ⅳ 5-hydroxydecannate (5-HD) and group Ⅴ 5-HD + Sevo. Focal cerebral I/R was produced by mid-cerebral artery occlusion (MCAO) in group Ⅱ-Ⅴ . Cerebral ischemia was maintained for 2 h followed by 6 and 24 h reporfnsiun. In group Ⅲ and Ⅴ 2.4% sevoflurane in 97.6% O_2 was inhaled for 60 min at 24 h before MCAO. In group Ⅴ 5-HD (a selective mito-K_(ATP) channel antagonist) 40 mg/kg was given intraporitoneally (IP) at 30 min before sevoflurane preconditioning. In group Ⅳ 100% O_2 was inhaled instead of sevoflurune and 5-HD 40 mg/kg was given IP at 30 min before O_2 inhalation. Neurological deficit score (NDS, 0=no deficit, 7 = death) were measured at 6 and 24 h of reperfusion and the animals were then killed. Their brains were removed for determination of infaret size by TTC and cellular translocation of PKCe by Western blot analysis. Results NDS were significantly lower and brain infarct size was significantly smaller in group.Sevo (Ⅲ) than in I/R group (Ⅱ). The neuroprotection induced by sevoflurane preconditioning was reversed by 5-HD 40 mg/kg in group Ⅴ (5-HD + Sevo). There was no significant difference in infarct size and NDS among group Ⅱ , Ⅳ and Ⅴ . PKCe was activated and translocated to the membrane fraction at 6 h of reperfusion in group Sevo (Ⅲ) and this effect of sevoflurane was also abolished by 5-HD in group Ⅴ (5-HD + Sevo). Conclusion Scvoflurane preconditioning-induced neuroprotection is mediated by mito-K_(ATP) channel probably through regulating PKCe membrane translocation. Key words: Anesthetics,inhalation; Ischemic preconditioning; KATP channels; Brain; Reperfusion injury

Role of protein kinase C in preconditioning-induced cerebral protection

Protein kinase C may play a central role in preconditioning-induced cerebral protection.Current findings confirm that some PKC isoforms involve in signaling pathways of mediating cerebral isehemic/reperfusion injury.Our previous studying results suggest that PKCe,γ involve in early stage of morphine preconditioning-induced cerebral ischemic tolerance.Further studies on the role of PKC in cerebral ischemic tolerance will provide scientific evidences for exploring new method of cerebral protection. Key words: Protein kinase C; Preconditioning; Cerebral isehemic tolerance