Pharmacological Protein Kinase C Modulators Reveal a Pro-hypertrophic Role for Novel Protein Kinase C Isoforms in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Hypertrophy of cardiomyocytes (CMs) is initially a compensatory mechanism to cardiac overload, but when prolonged, it leads to maladaptive myocardial remodeling, impairing cardiac function and causing heart failure. A key signaling molecule involved in cardiac hypertrophy is protein kinase C (PKC). However, the role of different PKC isoforms in mediating the hypertrophic response remains controversial. Both classical (cPKC) and novel (nPKC) isoforms have been suggested to play a critical role in rodents, whereas the role of PKC in hypertrophy of human CMs remains to be determined. Here, we aimed to investigate the effects of two different types of PKC activators, the isophthalate derivative HMI-1b11 and bryostatin-1, on CM hypertrophy and to elucidate the role of cPKCs and nPKCs in endothelin-1 (ET-1)-induced hypertrophy in vitro.Methods and Results: We used neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study the effects of pharmacological PKC modulators and ET-1. We used quantitative reverse transcription PCR to quantify hypertrophic gene expression and high-content analysis (HCA) to investigate CM morphology. In both cell types, ET-1, PKC activation (bryostatin-1 and HMI-1b11) and inhibition of cPKCs (Gö6976) increased hypertrophic gene expression. In NRVMs, these treatments also induced a hypertrophic phenotype as measured by increased recognition, intensity and area of α-actinin and F-actin fibers. Inhibition of all PKC isoforms with Gö6983 inhibited PKC agonist-induced hypertrophy, but could not fully block ET-1-induced hypertrophy. The mitogen-activated kinase kinase 1/2 inhibitor U0126 inhibited PKC agonist-induced hypertrophy fully and ET-1-induced hypertrophy partially. While ET-1 induced a clear increase in the percentage of pro-B-type natriuretic peptide-positive hiPSC-CMs, none of the phenotypic parameters used in HCA directly correlated with gene expression changes or with phenotypic changes observed in NRVMs.Conclusion: This work shows similar hypertrophic responses to PKC modulators in NRVMs and hiPSC-CMs. Pharmacological PKC activation induces CM hypertrophy via activation of novel PKC isoforms. This pro-hypertrophic effect of PKC activators should be considered when developing PKC-targeted compounds for e.g. cancer or Alzheimer’s disease. Furthermore, this study provides further evidence on distinct PKC-independent mechanisms of ET-1-induced hypertrophy both in NRVMs and hiPSC-CMs.

Download Full-text

Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl–Expressing UT-7 Cells

Blood ◽

10.1182/blood.v91.3.813.813_813_822 ◽

1998 ◽

Vol 91 (3) ◽

pp. 813-822 ◽

Cited By ~ 3

Author(s):

Ying Hong ◽

Dominique Dumènil ◽

Bernd van der Loo ◽

Frédérique Goncalves ◽

William Vainchenker ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Subcellular Distribution ◽

Membrane Fraction ◽

Induced Expression ◽

Gm Csf ◽

Kinase C ◽

Pkc Isoforms ◽

Mitogenic Action ◽

Pkc Activation

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms α and β found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms α and β to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

Download Full-text

PDGF-induced mitogenic signaling is not mediated through protein kinase C and c-fos pathway in VSM cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.1.c71 ◽

1993 ◽

Vol 264 (1) ◽

pp. C71-C79 ◽

Cited By ~ 59

Author(s):

R. V. Sharma ◽

R. C. Bhalla

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Dna Synthesis ◽

Thymidine Incorporation ◽

Cytosolic Calcium ◽

Platelet Derived Growth Factor ◽

Kinase C ◽

Pdgf Stimulation ◽

Pkc Activation

This study examines the role of protein kinase C (PKC) in platelet-derived growth factor (PDGF)-induced vascular smooth muscle (VSM) cell proliferation and initial signaling events. A 24-h pretreatment of VSM cells with 200 nM phorbol 12-myristate 13-acetate (PMA) completely abolished immunologically reactive PKC activity. Depletion of PKC activity from VSM cells did not attenuate PDGF-stimulated [3H]thymidine incorporation compared with control cells. Similarly, acute activation of PKC by treatment with 200 nM PMA for 10 min had no effect on PDGF-mediated [3H]thymidine incorporation. Both PMA and PDGF increased c-fos induction to the same magnitude; however, treatment with PMA did not induce DNA synthesis in these cells. In PKC-depleted cells PDGF-mediated c-fos induction was reduced by 50-60%, while DNA synthesis in response to PDGF stimulation was not reduced. PKC depletion did not alter PDGF-stimulated increase in cytosolic calcium levels, 125I-PDGF binding, or receptor autophosphorylation. On the basis of these results, we conclude that PKC activation and c-fos induction do not play a significant role in PDGF-mediated mitogenesis in VSM cells.

Download Full-text

Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Thromboxane Generation by Regulating cPLA2 Activation and Calcium Mobilization In Platelets

Blood ◽

10.1182/blood.v116.21.2020.2020 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2020-2020

Author(s):

Yamini Bynagari ◽

Parth Lakhani ◽

Kamala Bhavaraju ◽

Jianguo Jin ◽

Mario C Rico ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Conflicts Of Interest ◽

Arterial Injury ◽

Calcium Mobilization ◽

Erk Activation ◽

Kinase C ◽

Cpla2 Activation ◽

Pkc Isoforms

Abstract Abstract 2020 Positive regulatory role of Protein Kinase C (PKC) isoforms in platelets have been extensively studied. However, negative regulatory roles of PKCs in platelets are poorly understood. In this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms involved in this process. Pan PKC inhibition with GF109203X potentiated ADP-induced cPLA2 phosphorylation suggesting that PKCs negatively regulate thromboxane generation by regulating cPLA2 activation. Inhibition of PKCs potentiated ADP-induced ERK activation and intracellular calcium mobilization, two upstream signaling molecules of cPLA2.These data suggest that PKCs negatively regulate thromboxane by regulating ERK activation and calcium mobilization, which inturn regulate cPLA2 activation. Pan-PKC inhibition potentiated ADP-induced, P2Y1 receptor-mediated calcium mobilization in platelets independent of P2Y12-receptor. Pretreatment of platelets with GF109203X, a Pan PKC inhibitor, but not Go-6976, a classical PKC isoform inhibitor, potentiated ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We have previously demonstrated that nPKC η, θ, δ positively regulates agonist-induced thromboxane generation in platelets. Thus, we investigated if the role of nPKC ε in ADP-induced thromboxane generation using PKC ε knockout mice (PKCε KO). ADP-induced thromboxane generation in PKC ε KO murine platelets was ten-fold higher than that of wild type platelets. Furthermore, PKC ε KO mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in Tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and thereby offers protection against thrombosis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Vitamin E activates CRABP-II gene expression in cultured human fibroblasts, role of protein kinase C

FEBS Letters ◽

10.1016/j.febslet.2004.05.073 ◽

2004 ◽

Vol 569 (1-3) ◽

pp. 240-244 ◽

Cited By ~ 12

Author(s):

Amparo Gimeno ◽

Rosa Zaragozá ◽

Juan R Viña ◽

Vicente J Miralles

Keyword(s):

Gene Expression ◽

Protein Kinase C ◽

Vitamin E ◽

Protein Kinase ◽

Human Fibroblasts ◽

Kinase C ◽

Cultured Human Fibroblasts ◽

Crabp Ii

Download Full-text

Role of Protein Kinase C Isoforms in the Regulation of Interleukin-13-induced 15-Lipoxygenase Gene Expression in Human Monocytes

Journal of Biological Chemistry ◽

10.1074/jbc.m400413200 ◽

2004 ◽

Vol 279 (16) ◽

pp. 15954-15960 ◽

Cited By ~ 23

Author(s):

Bo Xu ◽

Ashish Bhattacharjee ◽

Biswajit Roy ◽

Gerald M. Feldman ◽

Martha K. Cathcart

Keyword(s):

Gene Expression ◽

Protein Kinase C ◽

Protein Kinase ◽

Human Monocytes ◽

Interleukin 13 ◽

Kinase C ◽

Lipoxygenase Gene ◽

Protein Kinase C Isoforms

Download Full-text

Protein kinase C regulates endocytosis and recycling of E-cadherin

AJP Cell Physiology ◽

10.1152/ajpcell.00566.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C489-C499 ◽

Cited By ~ 85

Author(s):

Tam Luan Le ◽

Shannon R. Joseph ◽

Alpha S. Yap ◽

Jennifer L. Stow

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Esters ◽

Recycling Endosomes ◽

Kinase C ◽

Pkc Isozymes ◽

Pkc Activation ◽

Darby Canine Kidney ◽

E Cadherin

E-cadherin is a major component of adherens junctions in epithelial cells. We showed previously that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. In the present study, we investigated the potential role of protein kinase C (PKC) in regulating the trafficking of surface E-cadherin in Madin-Darby canine kidney cells. Using surface biotinylation and immunofluorescence, we found that treatment of cells with phorbol esters increased the rate of endocytosis of E-cadherin, resulting in accumulation of E-cadherin in apically localized early or recycling endosomes. The recycling of E-cadherin back to the surface was also decreased in the presence of phorbol esters. Phorbol ester-induced endocytosis of E-cadherin was blocked by specific inhibitors, implicating novel PKC isozymes, such as PKC-ε in this pathway. PKC activation led to changes in the actin cytoskeleton facilitating E-cadherin endocytosis. Depolymerization of actin increased endocytosis of E-cadherin, whereas the PKC-induced uptake of E-cadherin was blocked by the actin stabilizer jasplakinolide. Our findings show that PKC regulates vital steps of E-cadherin trafficking, its endocytosis, and its recycling.

Download Full-text

Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon alpha

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1992.tb17352.x ◽

1992 ◽

Vol 209 (3) ◽

pp. 813-822 ◽

Cited By ~ 6

Author(s):

Robert I. JAMES ◽

Juan MENAYA ◽

Kati HUDSON ◽

Vinod DEVALIA ◽

Jonathan RYVES ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Protein Kinase C ◽

Protein Kinase ◽

Interferon Alpha ◽

Kinase C

Download Full-text

Phorbol 12,13-Diacetate-Induced Contraction of the Canine Basilar Artery: Role of Protein Kinase C

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1991.16 ◽

1991 ◽

Vol 11 (1) ◽

pp. 135-142 ◽

Cited By ~ 44

Author(s):

Makoto Sugawa ◽

Tohru Koide ◽

Shigetaka Naitoh ◽

Michiaki Takato ◽

Tohru Matsui ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Normal Medium ◽

Kinase C ◽

Prostaglandin F ◽

Basilar Arteries ◽

Canine Basilar Artery ◽

Biochemical Mechanisms ◽

Pkc Activation

The pharmacological and biochemical mechanisms of contractile responses to the protein kinase C (PKC) activator phorbol-12, 13-diacetate (PDA) were investigated in canine basilar arteries, In the normal medium, PDA elicited a strong, dose-related, and slow-developing sustained contraction, Among the constrictors examined, including serotonin, prostaglandin F2α and endothelin, only PDA yielded contractions in a 2 Ca2+ -free medium, In both media, the PDA-induced contractions were virtually inhibited by either staurosporine, H-7, or quinacrine, while neither neurotransmitter blockades nor R24571 (calmidazolium) exerted significant effects, In addition, it was shown that 8-bromocyclic GMP, but not 8-bromocyclic AMP, markedly curtailed the PDA-induced contractions, Biochemical analysis, furthermore, showed that PDA induced increased phosphorylations of 27- and 96-kDa and proteins other than the myosin light chain (MLC) 20-kDa protein, Thus, the present results open up a novel mechanism of sustained cerebral artery contractions, where PKC activation rather than Ca2+/calmodulin/MLC system plays a key role that is regulated both by phospholipase A 2 and by cyclic GMP.

Download Full-text

Bryostatin 1 activates protein kinase C and induces monocytic differentiation of HL-60 cells

Blood ◽

10.1182/blood.v72.1.208.bloodjournal721208 ◽

1988 ◽

Vol 72 (1) ◽

pp. 208-213 ◽

Cited By ~ 2

Author(s):

RM Stone ◽

E Sariban ◽

GR Pettit ◽

DW Kufe

Keyword(s):

Gene Expression ◽

Protein Kinase C ◽

Protein Kinase ◽

Growth Inhibition ◽

Phorbol Ester ◽

Phorbol Esters ◽

Cell Protein ◽

Monocytic Differentiation ◽

Bryostatin 1 ◽

Kinase C

Phorbol esters induce the human HL-60 promyelocytic cell line to differentiate along a monocytic pathway. This induction of differentiation may involve phorbol ester-induced activation of the phospholipid- and calcium-dependent protein kinase C. Bryostatin 1, a macrocyclic lactone, has been shown to compete with phorbol esters for binding to protein kinase C. We have confirmed that bryostatin 1 translocates activity of protein kinase C from the cytosolic to membrane fractions of HL-60 cells. The present results also demonstrate that bryostatin 1 (10 nmol/L) induces monocytic differentiation of HL- 60 cells as determined by adherence, growth inhibition, appearance of monocyte cell surface antigens, and alpha-naphthyl acetate esterase staining. Furthermore, bryostatin 1 (10 nmol/L) downregulated c-myc expression and induced c-fos, c-fms, and tumor necrosis factor transcripts. These changes in gene expression induced by bryostatin 1 are similar to those associated with phorbol ester-induced monocytic differentiation of HL-60 cells. In contrast, exposure to a higher concentration of bryostatin 1 (100 nmol/L) had less of an effect on growth inhibition of HL-60 cells and changes in gene expression. Moreover, 100 nmol/L bryostatin 1 antagonized the cytostatic effects and adherence induced by phorbol esters. Our results thus suggest that bryostatin 1 activates HL-60 cell protein kinase C and that this effect is associated with induction of monocytic differentiation.

Download Full-text