Overexpression Of Protein Kinase C Research Articles

Protein kinase C activity modulates nuclear Lamin A/C dynamics in HeLa cells

The nuclear lamina serves important functions in the nucleus, providing structural support to the nuclear envelope and contributing to chromatin organization. The primary proteins that constitute the lamina are nuclear lamins whose functions are impacted by post-translational modifications, including phosphorylation by protein kinase C (PKC). While PKC-mediated lamin phosphorylation is important for nuclear envelope breakdown during mitosis, less is known about interphase roles for PKC in regulating nuclear structure. Here we show that overexpression of PKC ß, but not PKC α, increases the Lamin A/C mobile fraction in the nuclear envelope in HeLa cells without changing the overall structure of Lamin A/C and Lamin B1 within the nuclear lamina. Conversely, knockdown of PKC ß, but not PKC α, reduces the Lamin A/C mobile fraction. Thus, we demonstrate an isoform-specific role for PKC in regulating interphase Lamin A/C dynamics outside of mitosis.

In This Issue

HomeCirculation ResearchVol. 129, No. 12In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Dec 2021https://doi.org/10.1161/RES.0000000000000521Circulation Research. 2021;129:1083is related toMyofilament Phosphorylation in Stem Cell Treated Diastolic Heart FailureCardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesisis related toAlternative Mitophagy Protects the Heart Against Obesity-Associated CardiomyopathyCardiac Resident Macrophages Prevent Fibrosis and Stimulate Angiogenesis (p 1086)Resident and recruited macrophages play different roles in cardiac hypertrophy, report Revelo et al.Download figureDownload PowerPointHigh blood pressure is a leading cause of cardiac hypertrophy—an increase in the heart’s muscle mass. In the short term, this growth helps the heart deal with the pressure overload but, if the pressure persists, pathological hypertrophy occurs, including fibrosis and stiffening of the muscle, and can lead to heart failure. It has been suggested that infiltrating macrophages may tip the scales towards pathology despite resident macrophages promoting tissue repair. To examine the roles of macrophages in hypertrophy more closely, Revelo and colleagues specifically depleted heart-resident macrophages in mice with a carefully designed antibody treatment that left circulating macrophages unaffected. They discovered that while the test and control mice responded similarly to heart pressure overload for the first week, in the long-term, the test mice exhibited accelerated decline in heart function and more severe fibrosis. Furthermore, mice whose circulating macrophages were also depleted were protected from this fibrosis. This suggests that recruited macrophages do indeed counteract the actions of resident ones and thereby promote pathology. Understanding the role of immune cells in heart failure will ultimately inform future therapies, say the team.Alternative Mitophagy Protects the Heart Against Obesity-Associated Cardiomyopathy (p 1105)An alternative form of mitophagy protects mouse hearts from obesity-related cardiomyopathy, say Tong et al.Download figureDownload PowerPointPeople with obesity or diabetes are at increased risk of developing cardiomyopathy which can eventually lead to heart failure. One of the major pathological features of obesity-related cardiomyopathy at the cellular level is a decrease in mitochondrial function, likely explained by a concurrent decrease in canonical mitophagy—a process whereby dysfunctional mitochondrial are degraded. An alternative mitophagy mechanism mediated by the factors Ulk1 and Rab9 has recently been discovered, however, and now Tong and colleagues show that this alternative version is actually upregulated steadily over 24 weeks in mice given a chronic high fat diet. Canonical mitophagy, by contrast, ceased after approximately two months. The team showed that suppressing the alternative mitophagy—either by knockout of Ulk1 or expression of a Rab9 loss-of-function mutant—further exacerbated the high-fat diet induced cardiac dysfunction, while over-expression of Rab9 in mouse hearts increased the alternative mitophagy and protected the animals from cardiac dysfunction. Together the results suggest that pharmacologically boosting this Ulk/Rab9-mediated alternative mitophagy may be a treatment strategy for preventing obesity-related cardiomyopathy.Myofilament Phosphorylation in Stem Cell Treated Diastolic Heart Failure (p 1125)Soetkamp et al suggest PKC inhibition as a way to treat a certain type of heart failure.Download figureDownload PowerPointWeakness, fatigue and trouble breathing are among the symptoms experienced by someone suffering heart failure with preserved ejection fraction (HFpEF). The pathology of the condition includes hypertrophy, fibrosis and stiffening of the heart with hyperphosphorylation of the cells’ sarcomeric proteins. Because this hyperphosphorylation is a key contributor to HFpEF pathology and because cardiosphere-derived stem cells (CDCs) show promise as a HFpEF treatment, Soetkamp and colleagues investigated whether CDC treatment reduces phosphorylation levels in the heart. Sure enough, administering CDCs to rats with HFpEF decreased the associated protein hyperphosphorylation compared with that seen in untreated animals. Bioinformatics analysis, based on the observed phosphorylation patterns and mass spectrometry data from test heart samples, suggested Protein Kinase C (PKC) as prime suspect behind the hyperphosphorylation—a suspicion strengthened by observing phosphorylation patterns in heart cells after inhibition or over-expression of PKC. The authors thus suggest CDCs alleviate HFpEF in part by reversing PKC-induced phosphoryation, and that PKC inhibition may be a desirable alternative treatment strategy especially as it avoids regulatory issues associated with cell-based therapies. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMyofilament Phosphorylation in Stem Cell Treated Diastolic Heart FailureDaniel Soetkamp, et al. Circulation Research. 2021;129:1125-1140Cardiac Resident Macrophages Prevent Fibrosis and Stimulate AngiogenesisXavier S. Revelo, et al. Circulation Research. 2021;129:1086-1101Alternative Mitophagy Protects the Heart Against Obesity-Associated CardiomyopathyMingming Tong, et al. Circulation Research. 2021;129:1105-1121 December 3, 2021Vol 129, Issue 12 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000521 Originally publishedDecember 2, 2021 PDF download Advertisement

Hypertension induces compensatory left ventricular hypertrophy by a mechanism involving gap junction lateralization and overexpression of CD36, PKC and MMP-2.

Hypertension-induced left ventricular hypertrophy evolves initially as an adaptive response meant to minimize ventricular wall stress. The mechanisms involved in the preservation of the cardiac function during the “compensatory” phase of the left ventricular hypertrophy are still unclear. Therefore, we aimed at uncovering fine changes that aid the heart to cope with the increased stress in hypertension. Male golden Syrian hamsters were given NG-nitro-L-arginine methyl ester (L-NAME) for 16 weeks, and they became hypertensive (HT), developing left ventricular hypertrophy with no impaired contractility or fibrosis. As compared to age-matched control hamsters, the hypertrophied left ventricles in L-NAME-induced HT hamsters exhibited the following structural and molecular changes: (i) accumulation of lipid droplets (LDs) within cardiomyocytes and relocation of gap junctions to the lateral membrane of cardiomyocytes or close to mitochondria (revealed by electron microscopy); (ii) overexpression of the cluster of differentiation 36 (CD36) fatty acid transporter, protein kinase C (PKC), and matrix metalloproteinase-2 (MMP-2), enhanced activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway, and unchanged expression of the connexin 43 (Cx43) and N-cadherin junctional proteins (assessed by Western blot); (iii) increased protein carbonyl content, assessed with a 2,4-Dinitrophenylhydrazine (DNPH)-based spectrophotometric assay, indicative of an enhanced reactive oxygen species (ROS) production; and (iv) augmented MMP-2 activity (determined by gelatin zymography). These changes may participate in an orchestrated adaptive hypertrophic growth response that helps to maintain cardiac performance, in HT hamsters. Together, these findings could provide support for designing future strategies meant to prevent the transition from compensatory left ventricular hypertrophy to decompensated heart failure.

Phorbol 12-myristate 13-acetate promotes nuclear translocation of hepatic steroid response element binding protein-2

Sterol regulatory element-binding protein (SREBP)-2 is a pivotal transcriptional factor in cholesterol metabolism. Factors interfering with the proper functioning of SREBP-2 potentially alter plasma lipid profiles. Phorbol 12-myristate 13-acetate (PMA), which is a common protein kinase C (PKC) activator, was shown to promote the post-translational processing and nuclear translocation of SREBP-2 in hepatic cells in the current study. Following SREBP-2 translocation, the transcripts of its target genes HMGCR and LDLR were upregulated as demonstrated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Electrophoretic mobility shift assays (EMSA) also demonstrated an induced DNA-binding activity on the sterol response element (SRE) domain under PMA treatment. The increase of activated Srebp-2 without the concurrent induced mRNA expression was also observed in an animal model. As the expression of SREBP-2 was not increased by PMA, the activation of PKC was the focus of investigation. Specific PKC isozyme inhibition and overexpression supported that PKCβ was responsible for the promoting effect. Further studies showed that the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not 5′ adenosine monophosphate-activated protein kinase (AMPK), were the possible downstream signaling proteins of PKCβ. In conclusion, this study illustrated that PKCβ increased SREBP-2 nuclear translocation in a pathway mediated by MEK/ERK and JNK, rather than the one dictated by AMPK. These results revealed a novel signaling target of PKCβ in the liver cells.

Synergistic anticancer effect of exogenous wild-type p53 gene combined with 5-FU in human colon cancer resistant to 5-FU in vivo.

To investigate the anticancer effect of a recombinant adenovirus-mediated p53 (rAd-p53) combined with 5-fluorouracil (5-FU) in human colon cancer resistant to 5-FU in vivo and the mechanism of rAd-p53 in reversal of 5-FU resistance. Nude mice bearing human colon cancer SW480/5-FU (5-FU resistant) were randomly assigned to four groups (n = 25 each): control group, 5-FU group, rAd-p53 group, and rAd-p53 + 5-FU group. At 24 h, 48 h, 72 h, 120 h and 168 h after treatment, 5 mice were randomly selected from each group and sacrificed using an overdose of anesthetics. The tumors were removed and the protein expressions of p53, protein kinase C (PKC), permeability-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) (Western blot) and apoptosis (TUNEL) were determined. The area ratios of tumor cell apoptosis were larger in the rAd/p53 + 5-FU group than that in the control, 5-FU and rAd/p53 groups (P < 0.05), and were larger in the rAd/p53 group than that of the control group (P < 0.05) and the 5-FU group at more than 48 h (P < 0.05). The p53 expression was higher in the rAd/p53 and the rAd/p53 + 5-FU groups than that of the control and 5-FU groups (P < 0.05), and were higher in the rAd/p53 + 5-FU group than that of the rAd/p53 group (P < 0.05). Overexpression of PKC, P-gp and MRP1 was observed in the 5-FU and control groups. In the rAd/p53 + 5-FU group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups (P < 0.05), and the expression of PKC was lower than that of the control, 5-FU and rAd/p53 groups at more than 48 h (P < 0.05). In the rAd/p53 group, the expression of P-gp and MRP1 was lower that of the control and 5-FU groups at more than 48 h (P < 0.05), and the expression of PKC was lower than that of the control and 5-FU groups at more than 120 h (P < 0.05). 5-FU combined with rAd-p53 has a synergistic anticancer effect in SW480/5-FU (5-FU resistance), which contributes to reversal of 5-FU resistance.

Abstract 2138: RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma

Abstract Uveal melanoma (UM) is the most common intraocular malignancy in adults and no effective treatment options are available for metastatic disease. Over 80% of UM show mutations in the Gαq family members GNAQ and GNA11. MAP-kinase pathway activation in part mediated by protein kinase C (PKC) has been shown as one critical contributing factor to GNAQ-mediated oncogenesis. However PKC inhibition alone does not completely suppress MAPK signaling. A more refined understanding of the signaling cascade linking MAPK signaling to mutant GNAQ or GNA11 is required to develop more effective strategies for targeted therapy. Among more than 10 different PKC isoforms, we identified both PKC δ and PKC ϵ to be required and sufficient to activate MAPK pathway in GNAQ mutant melanomas by using siRNA mediated knock-down and co-transfection of GNAQQ209L and specific PKC isoform cDNAs. Overexpression of GNAQQ209L in 293FT cells increased Ras-GTP level and knock down of three Ras isoforms in GNAQ mutant uveal melanoma cell lines decreased MAPK signaling. Microarray analysis of 5 different GNAQ/11 mutant and 5 NRAS/BRAF-mutant melanoma cells revealed RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), ranks at the top of 487 differentially expressed genes between (cut off : p&amp;lt;0.05, fold change &amp;gt;2 or &amp;lt;-2). Quantitative RT-PCR and western blot confirmed markedly (&amp;gt;100 fold) elevated RasGRP3 levels in GNAQ/11 mutant melanoma cells, while other RasGEFs were not significantly altered. Knock down of RasGRP3 decreased MAPK signaling and proliferation in GNAQ mutant melanoma cells, while it no effect on BRAF mutant cells. Mutating the PKC phosphorylation site of RasGRP3 (T133) partially attenuated RasGRP3-mediated MAPK signaling. Overexpression of PKC δ and PKC ϵ increased RasGRP3 T133 phosphorylation but not PKC α and PKC ζ. While PKC inhibition completely abrogated RasGRP3 T133 phosphorylation, it did not fully suppress RasGRP3-mediated MAPK signaling, indicating that its activating effect only partly requires PKC. We found that RasGRP3 activation mediated by its diacylglycerol(DAG) binding C1 domain provides an independent conduit of activation, which is augmented by PKC-mediated phosphorylation at T133. DAG is a second messenger that is released by phospholipase C, a direct effector of activated GNAQ/11. Finally, we found that the markedly elevated RasGRP3 expression in GNAQ mutant melanoma cell lines is a direct consequence of the oncogenic signaling and is mediated by PKC δ and ϵ. Hence, our data identify RasGRP3 as a critical signaling node linking oncogenic signaling downstream of GNAQ/11 to the Ras/Raf/MEK/ERK pathway through three mechanisms: phosphorylation by PKC δ and ϵ, binding of the second messenger DAG, and by upregulation of its protein level as a consequence of oncogenic GNAQ-mediated activation of PKC δ and PKC ϵ. The findings nominate RasGRP3 as a possible therapeutic target for cancers driven by oncogenic GNAQ/11. Citation Format: Xu Chen, Qiuxia Wu, Philippe Depeille, Jeroen P. Roose, Boris C. Bastian. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2015-2138

Abstract 5203: Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression

Abstract PKC is a serine-threonine kinase family that controls malignant transformation and metastatic dissemination. ATRA is the main active metabolite of vitamin A. Some evidences indicate that PKCδ may regulate the expression of some retinoid acid (RA) dependent genes, and others indicate that retinoids could alter PKCα intracellular localization; both processes would lead to cell differentiation. In this work we have developed human (MDA-MB 231) and murine (LM3) cell models overexpressing PKCα or PKCδ, in order to determine whether PKC expression alters the sensitivity to retinoids treatment (ATRA). The effect of ATRA was studied in vitro, analyzing biological responses related to tumor growth. In LM3 cells, only PKCα overexpression was able to reduce in vitro population doubling time (PDT) as compared to control (PDT: 14,8±2,3 h vs 21,2±3,1 h for LM3-PKCα y LM3-Vector respectively). Moreover, these cells also responded to retinoid treatment with a significant delay in cell proliferation (PDT: 24,3±4,2 h vs. 14,8±2,3 h for LM3-PKCα ATRA treated or not respectively. In MDA-MB 231 derived cell lines, PKC overexpression as well as ATRA treatment have no effect on proliferative potential. No differences were observed on migratory and invasive capabilities either. Interestingly, in LM3, PKCδ overexpression induced an important increase in proteolytic enzymes secretion, which correlates with its major invasiveness, but this increase had no impact on in vivo metastatic dissemination. Only PKCα overexpression increased this parameter (lung nodes, median (range): 55 (20-75) vs 0 (0-10) for LM3-PKCα y LM3-Vector respectively). Contrary to LM3-PKCδ in MDA-PKCδ cells we could detect a decrease on proteolytic enzymes production and invasive capability. Moreover, colonies growing in Matrigel as 3D cultures showed a small and branched structures. Finally we studied whether the overexpression of α and δ PKC isoforms is able to alter the activity of retinoic acid responsive elements (RARE) through a reporter gene assay. On LM3 model, the constitutive expression of PKCδ highly increased RARE dependent activity. Surprisingly, in MDA-MB231 derived sublines, we could detect a significant increase on RARE activity when cells were treated with ATRA, Altogether, these results suggest that PKCα overexpression confers a more aggressive phenotype in LM3 model, but also makes these cells sensitive to ATRA effects. We could hypothesize, that the absence of response to ATRA treatment displayed by MDA-MB 231 sublines can be explained by their lack of RARβ, which is implied in AP-1 transrepression in response to ATRA. Among others phenomena, AP-1 is involved in cell proliferation and proteolytic enzymes production. Regarding the differences of response to PKCδ overexpression of both models, it has been reported that this PKC isoform has a differential role, pro or anti tumorigenic, depending on cellular context. Citation Format: Maria I. Diaz Bessone, D. E. Berardi, S. M. Cirigliano, C. Flumian, E. D. Bal de Kier Joffe, L. B. Todaro, A. J. Urtreger. Differential response to retinoid treatment in mouse and human mammary tumor cell lines with alterations in protein kinase C (PKC) expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5203. doi:10.1158/1538-7445.AM2015-5203

Arsenite Acutely Decreases Nitric Oxide Production via the ROS-Protein Phosphatase 1-Endothelial Nitric Oxide Synthase-Thr(497) Signaling Cascade.

Chronic (>24 h) exposure of arsenite, an environmental toxicant, has shown the decreased nitric oxide (NO) production in endothelial cells (EC) by decreasing endothelial NO synthase (eNOS) expression and/or its phosphorylation at serine 1179 (eNOS-Ser1179 in bovine sequence), which is associated with increased risk of vascular diseases. Here, we investigated the acute (<24 h) effect of arsenite on NO production using bovine aortic EC (BAEC). Arsenite acutely increased the phosphorylation of eNOS-Thr497, but not of eNOS-Ser116 or eNOS-Ser1179, which was accompanied by decreased NO production. The level of eNOS expression was unaltered under this condition. Treatment with arsenite also induced reactive oxygen species (ROS) production, and pretreatment with a ROS scavenger N-acetyl-L-cysteine (NAC) completely reversed the observed effect of arsenite on eNOS-Thr497 phosphorylation. Although protein kinase C (PKC) and protein phosphatase 1 (PP1) were reported to be involved in eNOS-Thr497 phosphorylation, treatment with PKC inhibitor, Ro318425, and overexpression of various PKC isoforms did not affect the arsenite-stimulated eNOS-Thr497 phosphorylation. In contrast, treatment with PP1 inhibitor, calyculin A, mimicked the observed effect of arsenite on eNOS-Thr497 phosphorylation. Lastly, we found decreased cellular PP1 activity in arsenite-treated cells, which was reversed by NAC. Overall, our study demonstrates firstly that arsenite acutely decreases NO production at least in part by increasing eNOS-Thr497 phosphorylation via ROS-PP1 signaling pathway, which provide the molecular mechanism underlying arsenite-induced increase in vascular disease.

Sustained PKCβII activity confers oncogenic properties in a phospholipase D‐ and mTOR‐dependent manner

Protein kinase C (PKC) is a family of serine/threonine kinases implicated in a variety of physiological processes. We have shown previously that sustained activation of the classical PKCα and PKCβII induces their phospholipase D (PLD)-dependent internalization and translocation to a subset of the recycling endosomes defined by the presence of PKC and PLD (the pericentrion), which results in significant differences in phosphorylation of PKC substrates. Here, we have investigated the biological consequences of sustained PKC activity and the involvement of PLD in this process. We find that sustained activation of PKC results in activation of the mammalian target of rapamycin (mTOR)/S6 kinase pathway in a PLD- and endocytosis-dependent manner, with both pharmacologic inhibitors and siRNA implicating the PLD2 isoform. Notably, dysregulated overexpression of PKCβII in A549 lung cancer cells was necessary for the enhanced proliferation and migration of these cancer cells. Inhibition of PKCβII with enzastaurin reduced A549 cell proliferation by >60% (48 h) and migration by >50%. These biological effects also required both PLD activity and mTOR function, with both the PLD inhibitor FIPI and rapamycin reducing cell growth by >50%. Reciprocally, forced overexpression of wild-type PKCβII, but not an F666D mutant that cannot interact with PLD, was sufficient to enhance cell growth and increase migration of noncancerous HEK cells; indeed, both properties were almost doubled when compared to vector control and PKC-F666D-overexpressing cells. Notably, this condition was also dependent on both PLD and mTOR activity. In summary, these data define a PKC-driven oncogenic signaling pathway that requires both PLD and mTOR, and suggest that inhibitors of PLD or mTOR would be beneficial in cancers where PKC overexpression is a contributing or driving factor.

Abstract 554: Differential response to retinoid treatment of normal and transformed murine mammary cells overexpressing protein kinase C (PKC) isoforms.

Abstract Some of the most promising signaling pathways for breast cancer treatment include the PKC family, involved in proliferation and apoptosis, and the retinoid system mainly involved in differentiation. In this work we have overexpressed α or δ PKC isoforms in two murine mammary models, a tumor-derived cell line (LM3) and a normal mammary gland-derived line (NMuMG), in order to analyze whether elevated expression of these kinases alter the sensitivity to retinoid treatment. The overexpression of α or δ PKC isozymes did not induce detectable morphological alterations either in LM3 or in NMuMG cell lines. In the transformed cell context, the overexpression of PKCα increased cell proliferation (population doubling time: 14.8±2.3 h vs 21.2±3.1 h in control cells p&amp;lt;0.05) and motility (wound coverage: 69.3±8.3% vs 41.2±5.1% in control cells p&amp;lt;0.05), alterations that could be associated with an aggressive in vivo tumor behavior. Interestingly, at the same time, PKCα sensitized LM3 cells to the effects of the All-Trans-Retinoic Acid (ATRA) as evidenced by a significant reduction in both parameters. On the contrary, PKCδ overexpression did not affect either LM3 cells in vitro behavior or their response to ATRA. However, through a RARE-luciferase gene reporter assay, we could determine that the overexpression of PKCδ increased the activity of the retinoic acid receptors. In the normal cell context (NMuMG cells), the overexpression of both PKCs did not induce alterations in the proliferative potential (population doubling time: 14.8±0.8 h) or cell motility (wound coverage: 37.8±1.6%). Moreover, the subsequent treatment with ATRA neither affected the above mentioned parameters. Upon othotopic inoculation into syngeneic mice, LM3-PKCα cells formed tumors with higher growth rate and metastatic potential than LM3-vector ones. In vivo treatment with a subcutaneous ATRA pellet reduced both the local tumor growth and the number of spontaneous lung metastases (Md [Range]: 9 [0-27] vs 55 [20-75] for LM3-PKCα treated or not respectively p&amp;lt;0.05). PKCδ overexpression did not affect in vivo LM3 behavior. Our results show that in an already transformed context, PKCα overexpression induced a more aggressive phenotype but also conferred sensitivity to the retinoid treatment. On the other side high PKCδ levels increased the activity of retinoid receptors but did not modulate biological cell responses to this compound. In a normal cell context, both PKC isoforms as well as the retinoid treatment are innocuous thus ensuring the safeness use of retinoids in patients with aggressive breast tumors associated with high PKCα expression. Citation Format: María I. Díaz Bessone, Damian E. Berardi, Stefano M. Cirigliano, Carolina Flumian, Elisa D. Bal de Kier Joffe, Laura B. Todaro, Alejandro J. Urtreger. Differential response to retinoid treatment of normal and transformed murine mammary cells overexpressing protein kinase C (PKC) isoforms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 554. doi:10.1158/1538-7445.AM2013-554

Protein Kinase C Overexpression Does Not Enhance Immune-Stimulatory Surface Markers of Vaccinia-Infected Dendritic Cells and DC Cell Lines

One of the shortcomings of vaccinia virus (VACV) as immunization vector is the down-regulation of HLA and costimulatory molecules in antigen presenting cells. To overcome this problem we investigated the use of protein kinase C (PKC) as immune stimulatory agent. Thus several classical and atypical PKCs were inserted into wild-type or attenuated VACV using recombination into the hemagglutinin gene and the expression driven by the VACV 7,5K-IE gene promoter. Recombinant constructs expressing PKC-alpha, -beta, -theta as well as wild-type, constitutive active or dominant negative PKC-zeta constructs were generated. Additional constructs expressing PKB/Akt1 and ICAM-1 were used for comparison. Immature and mature peripheral blood derived-dendritic cells (DC) as well as lymphoid cell lines capable of obtaining a DC-like phenotype upon mitogen stimulation were infected. Disappointingly, VACV-driven PKC overexpression did not significantly enhance expression of various activation markers or costimulatory molecules tested. Neither CD86 nor HLA-DR expression was upregulated and also no influence on the maturation of DC, as measured by DC-SIGN and CD83, was observed. However, VACV did not interfere with LPS induced up-regulation of CD83 and did not lead to substantial apoptosis of infected DC within the first 24 hours.

PKC signaling inhibits osteogenic differentiation through the regulation of Msx2 function

Protein kinase C (PKC) signaling regulates osteoblast differentiation, but little is known about its downstream effectors. We examined the effect of modulating PKC activity on osteogenic transcription factors and found that the protein level of Msx2 is affected. Msx2 is induced by osteogenic signals such as BMPs and it plays critical roles in bone formation and osteoblast differentiation. Here, we examined the role of PKC signaling in regulating the function of Msx2. We found that the inhibition of PKC signaling enhances osteogenic differentiation in BMP2-stimulated C2C12 cells. Treatment with inhibitors of PKC activity or overexpression of kinase-defective (KD), dominant-negative mutant PKC isoforms strongly reduced the level of Msx2 protein. Several PKC isoforms (α, β, δ, and ζ) interacted with Msx2, and PKCβ phosphorylated Msx2 at Thr135 and Thr141. Msx2 repressed the transcriptional activity of the osteogenic transcription factor Runx2, and this repression was relieved by inhibition of PKC activity or overexpression of the KD mutant PKC isoforms. In addition, PKC prolonged the half-life of Msx2 protein. These results suggest that PKC signaling modulates osteoblast differentiation, at least in part, through the regulation of Msx2.

Abstract 172: Utility of retinoid treatment in the reversion of the malignant phenotype of mammary tumors with alterations in the expression of protein kinase C (PKC) isoforms

Abstract Some of the most promising signaling pathways for breast cancer treatment include the PKC family, involved in proliferation and apoptosis, and the retinoid system mainly involved in differentiation. In this work we have developed a murine model of mammary adenocarcinoma-derived cells (LM3) that overexpress α or ≤ PKC isoforms in order to analyze whether elevated levels of these kinases alter cell sensitivity to retinoid treatment (ATRA). LM3 cells are poorly responsive to ATRA. The overexpression of PKCα induced alterations in LM3 in vitro behavior that could be associated with tumor progression. In this sense, PKCα increased proliferation and motility (wound coverage: 69.3±8.3% vs 41.2±5.1% in control cells p&amp;lt;0.05) of the transfected LM3 cells. Interestingly, at the same time PKCα sensitized LM3 cells to ATRA treatment as evidenced by a significant reduction in both parameters. Upon othotopic inoculation into syngeneic mice LM3-PKCα cells formed tumors with higher growth rate and metastatic potential than LM3-vector cells. In vivo treatment with an ATRA pellet reduced both the local tumor growth and the number of spontaneous lung metastases (Md [Range]: 9 [0-27] vs 55 [20-75] for LM3-PKCα treated or not respectively p&amp;lt;0.05). On the contrary PKC ≤ overexpression did not affect LM3 cells behavior either in vitro or in vivo. LM3 cells response to ATRA was not modulated by PKC∈ either. However, through a RARE-luciferase gene reporter assay, we could determine that the overexpression of PKC∈ increased the activity of the retinoic acid receptors. Our results show that PKCα overexpression induced a more aggressive phenotype but also conferred ATRA sensitivity to mammary tumor cells. On the other side high PKC∈ levels increased the activity of retinoid receptors but did not modulate biological cell responses to retinoids. We believe that the use of retinoids may be beneficial in patients with aggressive breast tumors associated with high PKCα expression levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 172. doi:1538-7445.AM2012-172

Contribution of individual PKC isoforms to breast cancer progression

The protein kinase C (PKC) family of serine/threonine kinases has been intensively studied in cancer since their discovery as major receptors for the tumor-promoting phorbol esters. The contribution of each individual PKC isozyme to malignant transformation is only partially understood, but it is clear that each PKC plays different role in cancer progression. PKC deregulation is a common phenomenon observed in breast cancer, and PKC expression and localization are usually dynamically regulated during mammary gland differentiation and involution. In fact, the overexpression of several PKCs has been reported in malignant human breast tissue and breast cancer cell lines. In this review, we summarize the knowledge available on the specific roles of PKC isoforms in the development, progression, and metastatic dissemination of mammary cancer. We also discuss the role of PKC isoforms as therapeutic targets, and their potential as markers for prognosis or treatment response.

Regulation of Prostate Cancer Cell Survival by Protein Kinase C∈ Involves Bad Phosphorylation and Modulation of the TNFα/JNK Pathway

Protein kinase Cepsilon (PKCepsilon), a diacyglycerol- and phorbol ester-responsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKCdelta-mediated release of death factors, the involvement of PKCepsilon in this response is not known. PKCepsilon depletion by RNAi or expression of a dominant negative kinase-dead PKCepsilon mutant potentiated the apoptotic response of PMA and sensitized LNCaP cells to the death receptor ligand TNFalpha. On the other hand, overexpression of PKCepsilon by adenoviral means protected LNCaP cells against apoptotic stimuli. Interestingly, PKCepsilon RNAi depletion significantly enhanced the release of TNFalpha in response to PMA and greatly potentiated JNK activation by this cytokine. Further mechanistic analysis revealed that PMA fails to promote phosphorylation of Bad in Ser(112) in PKCepsilon-depleted LNCaP cells, whereas PKCepsilon overexpression greatly enhanced Bad phosphorylation. This effect was independent of Akt, ERK, or p90Rsk, well established kinases for Ser(112) in Bad. Moreover, expression of a S112A-Bad mutant potentiated PMA-induced apoptosis. Finally, we found that upon activation PKCepsilon accumulated in mitochondrial fractions in LNCaP cells and that Bad was a substrate of PKCepsilon in vitro. Our results established that PKCepsilon modulates survival in prostate cancer cells via multiple pathways.

Protein kinase C inhibits binding of diacylglycerol kinase-ζ to the retinoblastoma protein

We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-ζ (DGKζ) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKζ that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKζ binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKζ-MARCKS phosphorylation-site domain also prevented DGKζ binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKζ and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCα isoform inhibits DGKζ binding to pRB, whereas dominant-negative PKCα neutralized this inhibition. PKCα activation thus prevents DGKζ regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.

A major role of PKC θ and NFκB in the regulation of hTERT in human T lymphocytes

Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most human cells. In this work, we examined the participation of protein kinase C (PKC) in the regulation of hTERT expression in human T lymphocytes. Transient expression assays using luciferase reporter plasmids containing hTERT promoter showed that overexpression of PKC θ, but not the other PKC isoforms, could activate the promoter activity of hTERT in resting T lymphocytes. Among the PKC θ-activated signalings, we presented evidence that the expression of hTERT is mediated through NFκB but not through MEK or c-Jun N-terminal kinase pathways. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays, however, did not detect an increased binding of NFκB to the hTERT promoter in the activated T cells, although an increased binding of cMyc and Sp1 was detected. Together with the observation that inhibition of NFκB eliminated the induction of cMyc in activated T cells, these results suggest that PKC θ-activated NFκB signaling regulates the expression of hTERT via cMyc in human T lymphocytes.

Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation

Members of the PAR-1/MARK kinase family play critical roles in polarity and cell cycle control and are regulated by 14-3-3 scaffolding proteins, as well as the LKB1 tumour suppressor kinase and atypical protein kinase C (PKC). In this study, we initially investigated the mechanism underlying the interaction of mammalian MARK3 with 14-3-3. We demonstrate that 14-3-3 binding to MARK3 is dependent on phosphorylation, and necessitates the phosphate-binding pocket of 14-3-3. We found that interaction with 14-3-3 was not mediated by the previously characterised MARK3 phosphorylation sites, which led us to identify 15 novel sites of phosphorylation. Single point mutation of these sites, as well as the previously identified LKB1-(T211) and the atypical PKC sites (T564/S619), did not disrupt 14-3-3 binding. However, a mutant in which all 17 phosphorylation sites had been converted to alanine residues (termed 17A-MARK3), was no longer able to bind 14-3-3. Wild-type MARK3 was present in both the cytoplasm and plasma membrane, whereas the 17A-MARK3 mutant was strikingly localised at the plasma membrane. We provide data indicating that the membrane localisation of MARK3 required a highly conserved C-terminal domain, which has been termed kinase-associated domain-1 (KA-1). We also show that dissociation of 14-3-3 from MARK3 did not affect catalytic activity, and that a MARK3 mutant, which could not interact with 14-3-3, was normally active. Finally, we establish that there are significant differences in the subcellular localisation of MARK isoforms, as well as in the impact that atypical PKC overexpression has on 14-3-3 binding and localisation. Collectively, these results indicate that 14-3-3 binding to MARK isoforms is mediated by multiple phosphorylation sites, and serves to anchor MARK isoforms in the cytoplasm.

New Potential Agents in Treating Diabetic Kidney Disease

Despite the worldwide epidemic of chronic kidney disease complicating diabetes mellitus, current therapies directed against nephroprogression are limited to angiotensin conversion or receptor blockade. Nonetheless, additional therapeutic possibilities are slowly emerging. The diversity of therapies currently in development reflects the pathogenic complexity of diabetic nephropathy. The three most important candidate drugs currently in development include a glycosaminoglycan, a protein kinase C (PKC) inhibitor and an inhibitor of advanced glycation. In targeting primary mechanisms by which hyperglycaemia contributes to diabetic complications, these drugs could provide risk reduction complementary to the partial reduction proven for ACE inhibitors and angiotensin II receptor antagonists (angiotensin receptor blockers). Glycosaminoglycans act to restore glycoproteins present in reduced amounts in the glomerular basement membrane and mesangium of diabetic animal models. Components of the drug sulodexide prevent pathological changes and proteinuria in diabetic rats. Reductions in albuminuria, a hallmark of early diabetic kidney disease, have been reported in initial human trials. In the US, a multicentre phase II study has been completed, with an interim analysis indicating reduction in urinary albumin losses. Pivotal phase II trials have begun in patients with type 2 diabetes. A second metabolic pathway of diabetic complications is overexpression of PKC. Several activators of this family of intracellular kinases have been identified and PKC activation may result in tissue damage through a variety of mechanisms. In animal models, the inhibitor ruboxistaurin reduces albuminuria, diabetic histological changes and kidney injury. Like sulodexide, drug development of ruboxistaurin has reached completion of a phase II evaluation with mixed results. The third metabolic target is the nonenzymatic formulation of advanced glycation end-products (AGEs) through well described biochemical pathways. Multiple pathways lead to AGE accumulation in tissues in diabetes and diverse AGE products are formed. AGE deposition has been implicated in animal models of diabetic nephropathy. The leading AGE inhibitor currently in development is pyridoxamine, which has multiple actions that inhibit glycation. Pyridoxamine is an efficient AGE inhibitor in experimental diabetes. A phase II study in diabetic patients with nephropathy reported mixed efficacy results and a favourable safety profile. Phase III evaluation of pyridoxamine has not begun. These three classes of potential therapies, if successfully developed, will confirm that diabetic kidney disease has entered the era of biochemical treatments.

Transcriptional Repression of Protein Kinase Cα via Sp1 by Wild Type p53 Is Involved in Inhibition of Multidrug Resistance 1 P-Glycoprotein Phosphorylation

The protein kinase C (PKC) family consists of serine/threonine protein kinases that play important roles in signal transduction, cell proliferation, and tumor formation. Recent studies found that PKCs are commonly overexpressed in human tumors, including soft tissue sarcoma (STS). Overexpression of PKCs contributes to invasion and migration of tumor cells and induction of angiogenesis. PKC can also phosphorylate the multidrug resistance (MDR) gene-encoded P-glycoprotein and induce MDR phenotype. Our previous studies showed that mutation of p53 enhanced STS metastasis and mediated the MDR phenotype. Restoring wild type (WT) p53 in STS cells containing mutant p53 sensitized the cells to chemotherapy. In the present study, we found that PKCalpha protein expression is inhibited by WT p53 partly due to reduced PKCalpha mRNA expression in STS cells, but p53 does not affect PKCalpha mRNA stability. Deletion and mutation analysis of the PKCalpha promoter fused to the luciferase reporter gene identified a Sp1 binding site (-244/-234) in the PKCalpha promoter that is required for p53-mediated inhibition of PKCalpha promoter activity. More importantly, PKCalpha phosphorylates and activates MDR1 P-glycoprotein, whereas inhibition of PKCalpha by p53 leads to decreased MDR1 phosphorylation in STS cells, which sensitizes STS cells to chemotherapeutic agents. These data indicate that WT p53 may resensitize STS to chemotherapeutic agents by reducing MDR1 phosphorylation via transcriptional repression of PKCalpha expression. Thus, molecular-based therapies targeting mutant p53 and PKCalpha may be an effective new strategy to improve chemotherapeutic efficacy in STS.