Publications by authors named "Michael Leitges"

205 Publications

Protein kinase C and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

Cell Rep 2021 Nov;37(8):110054

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.
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http://dx.doi.org/10.1016/j.celrep.2021.110054DOI Listing
November 2021

WNT6/ACC2-induced storage of triacylglycerols in macrophages is exploited by Mycobacterium tuberculosis.

J Clin Invest 2021 08;131(16)

Microbial Interface Biology, Research Center Borstel, Leibniz Lung Center, Borstel, Germany.

In view of emerging drug-resistant tuberculosis (TB), host-directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase 2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights, compared with treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrate that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding that opens new perspectives for host-directed adjunctive treatment of pulmonary TB.
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http://dx.doi.org/10.1172/JCI141833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363280PMC
August 2021

A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism.

Life Sci Alliance 2021 08 18;4(8). Epub 2021 Jun 18.

Rudolf Virchow Center, Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany

Members of the protein kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver of animals fed a high-fat diet. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified more than 300 putative targets of PKD3. Furthermore, biochemical analysis revealed that PKD3 regulates cAMP-dependent PKA activity, a master regulator of the hepatic response to glucagon and fasting. PKA regulates glucose, lipid, and amino acid metabolism in the liver, by targeting key enzymes in the respective processes. Among them the PKA targets phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine. Consistently, we showed that PKD3 is activated by glucagon and promotes glucose and tyrosine levels in hepatocytes. Therefore, our data indicate that PKD3 might play a role in the hepatic response to glucagon.
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http://dx.doi.org/10.26508/lsa.202000863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8321662PMC
August 2021

Protein kinase C-β-dependent changes in the glucose metabolism of bone marrow stromal cells of chronic lymphocytic leukemia.

Stem Cells 2021 06 17;39(6):819-830. Epub 2021 Feb 17.

Department of Medicine 5-Hematology and Oncology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany.

Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-β (PKCβ) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCβ on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCβ that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCβ results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCβ-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCβ and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.
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http://dx.doi.org/10.1002/stem.3352DOI Listing
June 2021

Dysfunctional EGFR and oxidative stress-induced PKD1 signaling drive formation of DCLK1+ pancreatic stem cells.

iScience 2021 Jan 5;24(1):102019. Epub 2021 Jan 5.

Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL 32224, USA.

Doublecortin-like kinase 1 (DCLK1)-positive pancreatic cancer stem cells develop at a precancerous stage and may contribute to the lack of efficacy of pancreatic cancer therapy. Although PanIN cells express oncogenic KRas and have an increased activity of epidermal growth factor receptor (EGFR), we demonstrate that, in DCLK1 PanIN cells, EGFR signaling is not propagated to the nucleus. Mimicking blockage of EGFR with erlotinib in PanIN organoid culture or in p48;Kras mice led to a significant increase in DCLK1 PanIN cells. As a mechanism of how EGFR inhibition leads to formation of DCLK1 cells, we identify an increase in hydrogen peroxide contributing to activation of Protein Kinase D1 (PKD1). Active PKD1 then drives stemness and abundance of DCLK1 cells in lesions. Our data suggest a signaling mechanism that leads to the development of DCLK1 pancreatic cancer stem cells, which can be exploited to target this population in potential therapeutic approaches.
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http://dx.doi.org/10.1016/j.isci.2020.102019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820128PMC
January 2021

Investigations of mouse models during tumorigenesis revealed essential but distinct in vivo functions among the PKC family.

Authors:
Michael Leitges

Adv Biol Regul 2020 12 19;78:100756. Epub 2020 Sep 19.

Division of BioMedical Sciences, Faculty of Medicine, Craig L. Dobbin Genetics Research Centre, Memorial University of Newfoundland, Health Science Centre, 300 Prince Philip Drive, St. John's, NL, A1B 3V6, Canada. Electronic address:

PKC isozymes have been put in place as oncoproteins since the discovery that they can function as receptors for potent tumor-promoting phorbol esters in the 1980s. Despite nearly two decades of research, a clear in vivo proof of that concept was missing. The availability of so-called knock out mouse lines of individual PKC genes provided a tool to investigate isozyme specific in vivo functions in the context of tumor initiation, development and progression. This review aims to provide a limited overview of how the application of these mouse lines in combination with a cancer mouse model helped to understand PKC's in vivo function during tumorigenesis. The focus of this review will be on skin, colon and lung cancer.
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http://dx.doi.org/10.1016/j.jbior.2020.100756DOI Listing
December 2020

Stromal cell protein kinase C-β inhibition enhances chemosensitivity in B cell malignancies and overcomes drug resistance.

Sci Transl Med 2020 01;12(526)

Wellcome Trust/MRC Cambridge Stem Cell Institute and Department of Haematology, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AH, UK.

Overcoming drug resistance remains a key challenge to cure patients with acute and chronic B cell malignancies. Here, we describe a stromal cell-autonomous signaling pathway, which contributes to drug resistance of malignant B cells. We show that protein kinase C (PKC)-β-dependent signals from bone marrow-derived stromal cells markedly decrease the efficacy of cytotoxic therapies. Conversely, small-molecule PKC-β inhibitors antagonize prosurvival signals from stromal cells and sensitize tumor cells to targeted and nontargeted chemotherapy, resulting in enhanced cytotoxicity and prolonged survival in vivo. Mechanistically, stromal PKC-β controls the expression of adhesion and matrix proteins, required for activation of phosphoinositide 3-kinases (PI3Ks) and the extracellular signal-regulated kinase (ERK)-mediated stabilization of B cell lymphoma-extra large (BCL-X) in tumor cells. Central to the stroma-mediated drug resistance is the PKC-β-dependent activation of transcription factor EB, regulating lysosome biogenesis and plasma membrane integrity. Stroma-directed therapies, enabled by direct inhibition of PKC-β, enhance the effectiveness of many antileukemic therapies.
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http://dx.doi.org/10.1126/scitranslmed.aax9340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116365PMC
January 2020

Loss-of-function phenotype of a PKCθ knockin mouse strain.

Cell Commun Signal 2019 11 6;17(1):141. Epub 2019 Nov 6.

Department for Pharmacology and Genetics, Medical University Innsbruck, Innsbruck, Austria.

Background: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line.

Methods: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry.

Results: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression.

Conclusion: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.
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http://dx.doi.org/10.1186/s12964-019-0466-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836476PMC
November 2019

The kinase PKD3 provides negative feedback on cholesterol and triglyceride synthesis by suppressing insulin signaling.

Sci Signal 2019 08 6;12(593). Epub 2019 Aug 6.

Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, 97080 Würzburg, Germany.

Hepatic activation of protein kinase C (PKC) isoforms by diacylglycerol (DAG) promotes insulin resistance and contributes to the development of type 2 diabetes (T2D). The closely related protein kinase D (PKD) isoforms act as effectors for DAG and PKC. Here, we showed that PKD3 was the predominant PKD isoform expressed in hepatocytes and was activated by lipid overload. PKD3 suppressed the activity of downstream insulin effectors including the kinase AKT and mechanistic target of rapamycin complex 1 and 2 (mTORC1 and mTORC2). Hepatic deletion of PKD3 in mice improved insulin-induced glucose tolerance. However, increased insulin signaling in the absence of PKD3 promoted lipogenesis mediated by SREBP (sterol regulatory element-binding protein) and consequently increased triglyceride and cholesterol content in the livers of PKD3-deficient mice fed a high-fat diet. Conversely, hepatic-specific overexpression of a constitutively active PKD3 mutant suppressed insulin-induced signaling and caused insulin resistance. Our results indicate that PKD3 provides feedback on hepatic lipid production and suppresses insulin signaling. Therefore, manipulation of PKD3 activity could be used to decrease hepatic lipid content or improve hepatic insulin sensitivity.
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http://dx.doi.org/10.1126/scisignal.aav9150DOI Listing
August 2019

Protein Kinase Cι and Wnt/β-Catenin Signaling: Alternative Pathways to Kras/Trp53-Driven Lung Adenocarcinoma.

Cancer Cell 2019 08 1;36(2):156-167.e7. Epub 2019 Aug 1.

Department of Cancer Biology, Mayo Clinic Florida, 4500 San Pablo Road, Griffin Cancer Research Building, Room 212, Jacksonville, FL 32224, USA. Electronic address:

We report that mouse LSL-Kras;Trp53 (KP)-mediated lung adenocarcinoma (LADC) tumorigenesis can proceed through both PKCι-dependent and PKCι-independent pathways. The predominant pathway involves PKCι-dependent transformation of bronchoalveolar stem cells (BASCs). However, KP mice harboring conditional knock out Prkci alleles (KPI mice) develop LADC tumors through PKCι-independent transformation of Axin2 alveolar type 2 (AT2) stem cells. Transformed growth of KPI, but not KP, tumors is blocked by Wnt pathway inhibition in vitro and in vivo. Furthermore, a KPI-derived genomic signature predicts sensitivity of human LADC cells to Wnt inhibition, and identifies a distinct subset of primary LADC tumors exhibiting a KPI-like genotype. Thus, LADC can develop through both PKCι-dependent and PKCι-independent pathways, resulting in tumors exhibiting distinct oncogenic signaling and pharmacologic vulnerabilities.
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http://dx.doi.org/10.1016/j.ccell.2019.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693680PMC
August 2019

Novel mutant mouse line emphasizes the importance of protein kinase C theta for CD4 T lymphocyte activation.

Cell Commun Signal 2019 05 28;17(1):56. Epub 2019 May 28.

Department for Pharmacology and Genetics, Medical University Innsbruck, Division of Translational Cell Genetics, Peter Mayr Str. 1a, A-6020, Innsbruck, Austria.

Background: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation.

Methods: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry.

Results: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4 T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4 T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2 mice show a phenotype similar to conventional PKCθ-deficient mice.

Conclusion: Taken together, PKCθ-E2 mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4 T lymphocytes.
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http://dx.doi.org/10.1186/s12964-019-0364-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537413PMC
May 2019

PKCα integrates spatiotemporally distinct Ca and autocrine BDNF signaling to facilitate synaptic plasticity.

Nat Neurosci 2018 08 16;21(8):1027-1037. Epub 2018 Jul 16.

Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA.

The protein kinase C (PKC) enzymes have long been established as critical for synaptic plasticity. However, it is unknown whether Ca-dependent PKC isozymes are activated in dendritic spines during plasticity and, if so, how this synaptic activity is encoded by PKC. Here, using newly developed, isozyme-specific sensors, we demonstrate that classical isozymes are activated to varying degrees and with distinct kinetics. PKCα is activated robustly and rapidly in stimulated spines and is the only isozyme required for structural plasticity. This specificity depends on a PDZ-binding motif present only in PKCα. The activation of PKCα during plasticity requires both NMDA receptor Ca flux and autocrine brain-derived neurotrophic factor (BDNF)-TrkB signaling, two pathways that differ vastly in their spatiotemporal scales of signaling. Our results suggest that, by integrating these signals, PKCα combines a measure of recent, nearby synaptic plasticity with local synaptic input, enabling complex cellular computations such as heterosynaptic facilitation of plasticity necessary for efficient hippocampus-dependent learning.
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http://dx.doi.org/10.1038/s41593-018-0184-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100743PMC
August 2018

Protein Kinase C-β Dictates B Cell Fate by Regulating Mitochondrial Remodeling, Metabolic Reprogramming, and Heme Biosynthesis.

Immunity 2018 06 5;48(6):1144-1159.e5. Epub 2018 Jun 5.

Lymphocyte Interaction Laboratory, The Francis Crick Institute, London NW1 1AT, UK; Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA. Electronic address:

PKCβ-null (Prkcb) mice are severely immunodeficient. Here we show that mice whose B cells lack PKCβ failed to form germinal centers and plasma cells, which undermined affinity maturation and antibody production in response to immunization. Moreover, these mice failed to develop plasma cells in response to viral infection. At the cellular level, we have shown that Prkcb B cells exhibited defective antigen polarization and mTORC1 signaling. While altered antigen polarization impaired antigen presentation and likely restricted the potential of GC development, defective mTORC1 signaling impaired metabolic reprogramming, mitochondrial remodeling, and heme biosynthesis in these cells, which altogether overwhelmingly opposed plasma cell differentiation. Taken together, our study reveals mechanistic insights into the function of PKCβ as a key regulator of B cell polarity and metabolic reprogramming that instructs B cell fate.
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http://dx.doi.org/10.1016/j.immuni.2018.04.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015119PMC
June 2018

Shared and independent functions of aPKCλ and Par3 in skin tumorigenesis.

Oncogene 2018 09 23;37(37):5136-5146. Epub 2018 May 23.

Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Köln, Germany.

The polarity proteins Par3 and aPKC are key regulators of processes altered in cancer. Par3/aPKC are thought to dynamically interact with Par6 but increasing evidence suggests that aPKC and Par3 also exert complex-independent functions. Whereas aPKCλ serves as tumor promotor, Par3 can either promote or suppress tumorigenesis. Here we asked whether and how Par3 and aPKCλ genetically interact to control two-stage skin carcinogenesis. Epidermal loss of Par3, aPKCλ, or both, strongly reduced tumor multiplicity and increased latency but inhibited invasion to similar extents, indicating that Par3 and aPKCλ function as a complex to promote tumorigenesis. Molecularly, Par3/aPKCλ cooperate to promote Akt, ERK and NF-κB signaling during tumor initiation to sustain growth, whereas aPKCλ dominates in promoting survival. In the inflammatory tumorigenesis phase Par3/aPKCλ cooperate to drive Stat3 activation and hyperproliferation. Unexpectedly, the reduced inflammatory signaling did not alter carcinogen-induced immune cell numbers but reduced IL-4 Receptor-positive stromal macrophage numbers in all mutant mice, suggesting that epidermal aPKCλ and Par3 promote a tumor-permissive environment. Importantly, aPKCλ also serves a distinct, carcinogen-independent role in controlling skin immune cell homeostasis. Collectively, our data demonstrates that Par3 and aPKCλ cooperate to promote skin tumor initiation and progression, likely through sustaining growth, survival, and inflammatory signaling.
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http://dx.doi.org/10.1038/s41388-018-0313-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137026PMC
September 2018

Atypical PKC, PKCλ/ι, activates β-secretase and increases Aβ and phospho-tau in mouse brain and isolated neuronal cells, and may link hyperinsulinemia and other aPKC activators to development of pathological and memory abnormalities in Alzheimer's disease.

Neurobiol Aging 2018 01 15;61:225-237. Epub 2017 Sep 15.

Internal Medicine and Research Services, James A. Haley Veterans Medical Center, Tampa, Florida, USA; Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida, USA. Electronic address:

Hyperinsulinemia activates brain Akt and PKC-λ/ι and increases Aβ and phospho-tau in insulin-resistant animals. Here, we examined underlying mechanisms in mice, neuronal cells, and mouse hippocampal slices. Like Aβ, β-secretase activity was increased in insulin-resistant mice and monkeys. In insulin-resistant mice, inhibition of hepatic PKC-λ/ι sufficient to correct hepatic abnormalities and hyperinsulinemia simultaneously reversed increases in Akt, atypical protein kinase C (aPKC), β-secretase, and Aβ, and restored acute Akt activation. However, 2 aPKC inhibitors additionally blocked insulin's ability to activate brain PKC-λ/ι and thereby increase β-secretase and Aβ. Furthermore, direct blockade of brain aPKC simultaneously corrected an impairment in novel object recognition in high-fat-fed insulin-resistant mice. In neuronal cells and/or mouse hippocampal slices, PKC-ι/λ activation by insulin, metformin, or expression of constitutive PKC-ι provoked increases in β-secretase, Aβ, and phospho-thr-231-tau that were blocked by various PKC-λ/ι inhibitors, but not by an Akt inhibitor. PKC-λ/ι provokes increases in brain β-secretase, Aβ, and phospho-thr-231-tau. Excessive signaling via PKC-λ/ι may link hyperinsulinemia and other PKC-λ/ι activators to pathological and functional abnormalities in Alzheimer's disease.
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http://dx.doi.org/10.1016/j.neurobiolaging.2017.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705272PMC
January 2018

Protein kinase Cι/λ is dispensable for platelet function in thrombosis and hemostasis in mice.

Cell Signal 2017 10 21;38:223-229. Epub 2017 Jul 21.

Institute of Experimental Biomedicine, University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Germany. Electronic address:

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or ischemic stroke. Upon platelet activation, cytoskeletal reorganization is essential for platelet secretion and thrombus formation. Members of the protein kinase C family, which includes 12 isoforms, are involved in most platelet responses required for thrombus formation. The atypical protein kinase Cι/λ (PKCι/λ) has been implicated as an important mediator of cell polarity, carcinogenesis and immune cell responses. PKCι/λ is known to be associated with the small GTPase Cdc42, an important mediator of multiple platelet functions; however, its exact function in platelets is not known. To study the role of PKCι/λ, we generated platelet- and megakaryocyte-specific PKCι/λ knockout mice (Prkci) and used them to investigate the function of PKCι/λ in platelet activation and aggregation in vitro and in vivo. Surprisingly, lack of PKCι/λ had no detectable effect on platelet spreading and function in vitro and in vivo under all tested conditions. These results indicate that PKCι/λ is dispensable for Cdc42-triggered processes and for thrombosis and hemostasis in mice.
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http://dx.doi.org/10.1016/j.cellsig.2017.07.015DOI Listing
October 2017

Deletion of Protein Kinase C λ in POMC Neurons Predisposes to Diet-Induced Obesity.

Diabetes 2017 04 10;66(4):920-934. Epub 2017 Jan 10.

UW Diabetes Institute and Department of Medicine, University of Washington, Seattle, WA

Effectors of the phosphoinositide 3-kinase (PI3K) signal transduction pathway contribute to the hypothalamic regulation of energy and glucose homeostasis in divergent ways. Here we show that central nervous system (CNS) action of the PI3K signaling intermediate atypical protein kinase C (aPKC) constrains food intake, weight gain, and glucose intolerance in both rats and mice. Pharmacological inhibition of CNS aPKC activity acutely increases food intake and worsens glucose tolerance in chow-fed rodents and causes excess weight gain during high-fat diet (HFD) feeding. Similarly, selective deletion of the aPKC isoform in proopiomelanocortin (POMC) neurons disrupts leptin action, reduces melanocortin content in the paraventricular nucleus, and markedly increases susceptibility to obesity, glucose intolerance, and insulin resistance specifically in HFD-fed male mice. These data implicate aPKC as a novel regulator of energy and glucose homeostasis downstream of the leptin-PI3K pathway in POMC neurons.
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http://dx.doi.org/10.2337/db16-0482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360303PMC
April 2017

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Cell Metab 2017 01 17;25(1):197-207. Epub 2016 Nov 17.

Department of Molecular Genetics, CARIM, Maastricht University, 6200 MD Maastricht, the Netherlands; Department of Pathology, CARIM, Maastricht University, 6200 MD Maastricht, the Netherlands. Electronic address:

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.
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http://dx.doi.org/10.1016/j.cmet.2016.10.013DOI Listing
January 2017

Role of Integrins α1β1 and α2β1 in Wound and Tumor Angiogenesis in Mice.

Am J Pathol 2016 11 14;186(11):3011-3027. Epub 2016 Sep 14.

Department of Dermatology, University of Cologne, Cologne, Germany. Electronic address:

Integrins are transmembrane receptors composed of one α subunit and one β subunit and are involved in cellular growth, differentiation, and apoptosis. The collagen-binding integrins α1β1 and α2β1 have been shown to regulate wound and tumor vascularization by different mechanisms. In this study, we assessed wound and tumor vascularization in mice with genetic ablation of both integrin subunits α1 and α2, which resulted in loss of integrins α1β1 and α2β1. Wound angiogenesis was investigated in excisional wounds that were inflicted on the back skin of control and mice lacking integrin α1β1 and α2β1. Mutant mice displayed reduced wound angiogenesis, which correlated with decreased macrophage numbers at 3 and 7 days after injury, and showed significantly attenuated vascularization of sponge implants. Angiogenesis induced by tumors arising from intradermal injection of B16 F1 melanoma cells was also reduced in comparison to controls 7 days after injection. This reduction in angiogenesis correlated with increased levels and activity of circulating matrix metalloproteinase 9 and elevated angiostatin levels in plasma of mutant mice, which reduced endothelial cell proliferation. Ex vivo mutant aortic ring explants developed significantly fewer and thinner aortic sprouts with fewer branch points than controls because of impaired endothelial cell proliferation. In conclusion, the loss of integrins α1β1 and α2β1 in mice results in reduced wound and tumor angiogenesis by cell-autonomous and extrinsic mechanisms.
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http://dx.doi.org/10.1016/j.ajpath.2016.06.021DOI Listing
November 2016

Disease Phenotypes in a Mouse Model of RNA Toxicity Are Independent of Protein Kinase Cα and Protein Kinase Cβ.

PLoS One 2016;11(9):e0163325. Epub 2016 Sep 22.

Department of Pathology, University of Virginia, Charlottesville, Virginia, United States of America.

Myotonic dystrophy type 1(DM1) is the prototype for diseases caused by RNA toxicity. RNAs from the mutant allele contain an expanded (CUG)n tract within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The toxic RNAs affect the function of RNA binding proteins leading to sequestration of muscleblind-like (MBNL) proteins and increased levels of CELF1 (CUGBP, Elav-like family member 1). The mechanism for increased CELF1 is not very clear. One favored proposition is hyper-phosphorylation of CELF1 by Protein Kinase C alpha (PKCα) leading to increased CELF1 stability. However, most of the evidence supporting a role for PKC-α relies on pharmacological inhibition of PKC. To further investigate the role of PKCs in the pathogenesis of RNA toxicity, we generated transgenic mice with RNA toxicity that lacked both the PKCα and PKCβ isoforms. We find that these mice show similar disease progression as mice wildtype for the PKC isoforms. Additionally, the expression of CELF1 is also not affected by deficiency of PKCα and PKCβ in these RNA toxicity mice. These data suggest that disease phenotypes of these RNA toxicity mice are independent of PKCα and PKCβ.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033491PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163325PLOS
September 2016

Control of Paneth Cell Fate, Intestinal Inflammation, and Tumorigenesis by PKCλ/ι.

Cell Rep 2016 09;16(12):3297-3310

Cancer Metabolism and Signaling Networks Program, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address:

Paneth cells are a highly specialized population of intestinal epithelial cells located in the crypt adjacent to Lgr5(+) stem cells, from which they differentiate through a process that requires downregulation of the Notch pathway. Their ability to store and release antimicrobial peptides protects the host from intestinal pathogens and controls intestinal inflammation. Here, we show that PKCλ/ι is required for Paneth cell differentiation at the level of Atoh1 and Gfi1, through the control of EZH2 stability by direct phosphorylation. The selective inactivation of PKCλ/ι in epithelial cells results in the loss of mature Paneth cells, increased apoptosis and inflammation, and enhanced tumorigenesis. Importantly, PKCλ/ι expression in human Paneth cells decreases with progression of Crohn's disease. Kaplan-Meier survival analysis of colorectal cancer (CRC) patients revealed that low PRKCI levels correlated with significantly worse patient survival rates. Therefore, PKCλ/ι is a negative regulator of intestinal inflammation and cancer through its role in Paneth cell homeostasis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043519PMC
http://dx.doi.org/10.1016/j.celrep.2016.08.054DOI Listing
September 2016

Novel Insights into the PKCβ-dependent Regulation of the Oxidoreductase p66Shc.

J Biol Chem 2016 Nov 13;291(45):23557-23568. Epub 2016 Sep 13.

From the Daniel Swarovski Research Laboratory, Department of Visceral, Transplant, and Thoracic Surgery,

Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield HO, which in turn initiates cell death. PKCβ-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCβ thus may provide a target for therapeutic intervention. We performed a reassessment of PKCβ regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser phosphorylation by PKCβ, they instead provided evidence for Ser and Ser as PKCβ phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCβ phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr and Ser mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCβ-dependent regulation of p66 activation involving Ser and a motif surrounding Ser.
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http://dx.doi.org/10.1074/jbc.M116.752766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095410PMC
November 2016

A bright future for protein kinase D1 as a drug target to prevent or treat pancreatic cancer.

Mol Cell Oncol 2016 Jan 10;3(1):e1035477. Epub 2015 Dec 10.

The Biotechnology Center of Oslo, University of Oslo , Oslo, Norway.

Pancreatic ductal adenocarcinoma originates from acinar cells that undergo acinar-to-ductal metaplasia (ADM). ADM is initiated in response to growth factors, inflammation, and oncogene activation and leads to a de-differentiated, duct-like phenotype. Our recent publication demonstrated a transforming growth factor α-Kras(G12D)-protein kinase D1-Notch1 signaling axis driving the induction of ADM and further progression to pancreatic intraepithelial neoplasia. This suggests that protein kinase D1 might be an early marker for tumor development and a potential target for drug development.
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http://dx.doi.org/10.1080/23723556.2015.1035477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845192PMC
January 2016

PKD3 deficiency causes alterations in microtubule dynamics during the cell cycle.

Cell Cycle 2016 07 31;15(14):1844-54. Epub 2016 May 31.

a Biotechnology Center of Oslo , University of Oslo , Oslo , Norway.

Protein kinase D 3 (PKD3) is a member of the PKD family that has been linked to many intracellular signaling pathways. However, defined statements regarding isoform specificity and in vivo functions are rare. Here, we use mouse embryonic fibroblast cells that are genetically depleted of PKD3 to identify isoform-specific functions. We show that PKD3 is involved in the regulation of the cell cycle by modulating microtubule nucleation and dynamics. In addition we also show that PKD1 partially can compensate for PKD3 function. Taken together our data provide new insights of a specific PKD3 signaling pathway by identifying a new function, which has not been identified before.
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http://dx.doi.org/10.1080/15384101.2016.1188237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968898PMC
July 2016

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer's disease.

Sci Signal 2016 05 10;9(427):ra47. Epub 2016 May 10.

Department of Neurosciences and Division of Biology, Section of Neurobiology, University of California, San Diego, La Jolla, CA 92093, USA.

Alzheimer's disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-β (Aβ) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme.
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http://dx.doi.org/10.1126/scisignal.aaf6209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154619PMC
May 2016

Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions.

Cell Rep 2016 Mar 3;14(10):2325-36. Epub 2016 Mar 3.

Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32224, USA. Electronic address:

The development of pancreatic cancer requires the acquisition of oncogenic KRas mutations and upregulation of growth factor signaling, but the relationship between these is not well established. Here, we show that mutant KRas alters mitochondrial metabolism in pancreatic acinar cells, resulting in increased generation of mitochondrial reactive oxygen species (mROS). Mitochondrial ROS then drives the dedifferentiation of acinar cells to a duct-like progenitor phenotype and progression to PanIN. This is mediated via the ROS-receptive kinase protein kinase D1 and the transcription factors NF-κB1 and NF-κB2, which upregulate expression of the epidermal growth factor, its ligands, and their sheddase ADAM17. In vivo, interception of KRas-mediated generation of mROS reduced the formation of pre-neoplastic lesions. Hence, our data provide insight into how oncogenic KRas interacts with growth factor signaling to induce the formation of pancreatic cancer.
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http://dx.doi.org/10.1016/j.celrep.2016.02.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794374PMC
March 2016

Brain Insulin Signaling Is Increased in Insulin-Resistant States and Decreases in FOXOs and PGC-1α and Increases in Aβ1-40/42 and Phospho-Tau May Abet Alzheimer Development.

Diabetes 2016 07 19;65(7):1892-903. Epub 2016 Feb 19.

Medical, Neurology, Psychiatry, and Research Services, James A. Haley Veterans Hospital, Tampa, FL Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL

Increased coexistence of Alzheimer disease (AD) and type 2 diabetes mellitus (T2DM) suggests that insulin resistance abets neurodegenerative processes, but linkage mechanisms are obscure. Here, we examined insulin signaling factors in brains of insulin-resistant high-fat-fed mice, ob/ob mice, mice with genetically impaired muscle glucose transport, and monkeys with diet-dependent long-standing obesity/T2DM. In each model, the resting/basal activities of insulin-regulated brain protein kinases, Akt and atypical protein kinase C (aPKC), were maximally increased. Moreover, Akt hyperactivation was accompanied by hyperphosphorylation of substrates glycogen synthase kinase-3β and mammalian target of rapamycin and FOXO proteins FOXO1, FOXO3A, and FOXO4 and decreased peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression. Akt hyperactivation was confirmed in individual neurons of anterocortical and hippocampal regions that house cognition/memory centers. Remarkably, β-amyloid (Aβ1-40/42) peptide levels were as follows: increased in the short term by insulin in normal mice, increased basally in insulin-resistant mice and monkeys, and accompanied by diminished amyloid precursor protein in monkeys. Phosphorylated tau levels were increased in ob/ob mice and T2DM monkeys. Importantly, with correction of hyperinsulinemia by inhibition of hepatic aPKC and improvement in systemic insulin resistance, brain insulin signaling normalized. As FOXOs and PGC-1α are essential for memory and long-term neuronal function and regeneration and as Aβ1-40/42 and phospho-tau may increase interneuronal plaques and intraneuronal tangles, presently observed aberrations in hyperinsulinemic states may participate in linking insulin resistance to AD.
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http://dx.doi.org/10.2337/db15-1428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915579PMC
July 2016
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