DOCSLIB.ORG

Regulation of Matrix Gla Protein by Parathyroid Hormone in MC3T3-E1 Osteoblast-Like Cells Involves Protein Kinase a and Extracellular Signal-Regulated Kinase Pathways

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Matrix Gla Protein by Parathyroid Hormone in MC3T3-E1 Osteoblast-Like Cells Involves Protein Kinase a and Extracellular Signal-Regulated Kinase Pathways Journal of Cellular Biochemistry 102:496–505 (2007) Regulation of Matrix Gla Protein by Parathyroid Hormone in MC3T3-E1 Osteoblast-Like Cells Involves Protein Kinase A and Extracellular Signal-Regulated Kinase Pathways Supaporn Suttamanatwong,1 Renny T. Franceschi,2 Ann E. Carlson,1 and Rajaram Gopalakrishnan1* 1Department of Diagnostic and Biological Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota 55455 2Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109 Abstract Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix g-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a À748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 À7 M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co- transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways. J. Cell. Biochem. 102: 496–505, 2007. ß 2007 Wiley-Liss, Inc. Key words: parathyroid hormone; matrix gla protein; protein kinase A; protein kinase C; extracellular signal-related kinase; osteoblast Parathyroid hormone (PTH) functions as a can be either anabolic or catabolic, depending on key regulator of calcium homeostasis by acting dosage, method of delivery, and frequency of through its target organs, that is, bone, kidney, treatment [Tam et al., 1982]. Intermittent low and intestine, to increase serum calcium levels. dose administration of PTH in vivo promotes In bone, PTH serves as a major mediator of bone bone formation while continuous treatment remodeling. The in vivo effects of PTH on bone results in bone loss [Ishizuya et al., 1997; Jilka et al., 1999]. Continuous exposure to PTH in vitro has been shown to inhibit osteoblast differentiation and mineralization in primary calvarial osteoblasts, calvarial explants, and Grant sponsor: NIH; Grant number: DE016093; Grant osteoblast-like cell lines [Bellows et al., 1990; sponsor: University of Minnesota. Pfeilschifter et al., 1990; Ishizuya et al., 1997; *Correspondence to: Rajaram Gopalakrishnan, BDS, PhD, Koh et al., 1999; Wang et al., 2000]. Department of Diagnostic and Biological Sciences, Uni- versity of Minnesota School of Dentistry, 16-108B Moos In osteoblasts, actions of PTH are mediated by Tower, 515 Delaware St. SE, Minneapolis, MN 55455, USA the PTH1 receptor (PTH1R), a seven transmem- E-mail: [email protected] brane domain G-protein coupled receptor Received 2 October 2006; Accepted 30 January 2007 expressed on the cell surface. PTH treatment DOI 10.1002/jcb.21314 is known to stimulate three major signaling ß 2007 Wiley-Liss, Inc. Signaling Pathways in PTH Regulation of MGP 497 cascades leading to activation of protein kinase osteoblast-like cells express MGP, and PTH A (PKA), protein kinase C (PKC), and extra- treatment induces MGP mRNA level in a time- cellular signal-regulated kinase (ERK) path- and dose-dependent manner [Gopalakrishnan ways [Partridge et al., 1994; Swarthout et al., et al., 2001]. We also showed that MGP is a key 2002]. Following activation of these signaling regulator in PTH-mediated inhibition of miner- pathways, PTH regulates its target genes alization in these cells. To further understand by activation of several transcription factors the mechanism by which PTH regulates MGP [Partridge et al., 1994]. The cAMP response expression in osteoblasts, we investigated the element binding protein (CREB), activator signaling pathways involved in PTH regulation protein-1 (AP-1) transcription factors, and of MGP mRNA level and promoter activity in RUNX2 are among the most widely studied MC3T3-E1 cells. Our studies show that actions PTH-responsive transcription factors in osteo- of PTH on MGP can be explained, at least in blasts. Post-translational modifications such part, by stimulation of MGP promoter activity. as phosphorylation are required for activation Furthermore, this activation is mediated by of these transcription factors, which in turn both PKA- and ERK/MAPK pathways. regulate changes in gene expression. Matrix g-carboxyglutamic acid (Gla) protein EXPERIMENTAL PROCEDURES (MGP) is a vitamin K-dependent protein found DNA Constructs in hypertrophic chondrocytes, cementum, vas- cular smooth muscle cells, and bone [Price, The murine Mgp promoter construct, pMGP- 1989; Sato et al., 1998]. MGP is produced by a luc, containing bases À748 to þ1 of the murine variety of cell types in culture, including osteo- Mgp gene was derived from pMGP2 and blasts, chondrocytes, vascular smooth muscle subcloned into the HindIII site of pGL3Basic cells, and endothelial cells [Fraser et al., 1988; (Promega, Madison, WI) upstream of the firefly Barone et al., 1991; Engelse et al., 2001; luciferase reporter gene. The plasmid pMGP2 Gopalakrishnan et al., 2001]. MGP functions was a gift from Dr.Gerard Karsenty (Baylor as an inhibitor of mineralization both in vitro University, Houston, TX) [Luo et al., 1995]. and in vivo [Luo et al., 1997]. Retrovirus- pCMV5-MEK(SP), a plasmid encoding consti- mediated overexpression of MGP greatly tutively active mutant of MEK, was kindly decreased mineralization in cultured chondro- provided by Dr. Kun-Liang Guan (University of cytes and inhibited cartilage mineralization as Michigan, Ann Arbor, MI) and was used as well as endochondral ossification in developing described previously [Xiao et al., 2000]. chick limb bud [Yagami et al., 1999]. Major Cell Culture in vivo evidence that further substantiates the role of MGP as a potent mineralization inhibitor A highly differentiating subclone of MC3T3- comes from studies in MGP-deficient mice. E1 cells (MC-14) was used for the studies [Wang These mice exhibit abnormal calcification of et al., 1999; Petryk et al., 2005]. MC3T3-E1 cells cartilage and extensive mineralization of arteri- express most of the known osteoblast markers es [Luo et al., 1997]. Mutation of the MGP gene and form a mineralized ECM following differ- in humans results in Keutel syndrome, an entiation in AA-containing medium. MC3T3-E1 autosomal recessive disorder characterized by cells were maintained in MEM media contain- abnormal cartilage calcification, peripheral ing 10% FBS, 1% penicillin/streptomycin, 1% pulmonary stenosis, and midfacial hypoplasia non-essential amino acids, and were not used [Munroe et al., 1999]. beyond passage 20. For all experiments except Although several studies have been pub- those involving transfection, cells were plated lished, investigating the regulation of MGP at 50,000 cells/cm2 and treatments performed in expression in chondrocytes, vascular smooth 0.1% FBS containing MEM. Experiments invol- muscle cells, and endothelial cells by several ving signaling inhibitors were pretreated for growth factors and hormones such as retinoic 30 min with signaling inhibitors. The following acid, TGFb, FGF2, and thyroid hormone specific activators and inhibitors for each [Cancela and Price, 1992; Kirfel et al., 1997; signaling pathway were used; PKA inhibitor Stheneur et al., 2003; Sato et al., 2005], not much (H-89); PKC inhibitors (GF109203X); MEK/ is known about regulation of MGP in osteo- ERK inhibitors (U0126); PKA activators blasts. Previously, we showed that MC3T3-E1 (forskolin and 8-Br cAMP); and PKC activator 498 Suttamanatwong et al. [phorbol 12-myristate 13-acetate (PMA)]. For Real-Time RT-PCR experiments involving transfection, MC3T3-E1 Reverse transcription of extracted mRNA cells were plated at a density of 25,000 cells/cm2 was performed using 1 mg of denatured RNA in 35-mm dishes. and 100 pmol of random hexamers (Invitrogen Western Blot Analysis Life Technologies) in a 20 ml total volume containing 50 U Super Script reverse transcrip- MC3T3-E1 cells were serum starved for 24 h, tase (Invitrogen Life Technologies). Quantita- following which cells were pretreated with tive RT-PCR was performed as previously the indicated concentration of GF109203X or described using MX MX 3000PTM real-time U0126 for 30 min in AA-free MEM containing PCR System (Stratagene Corporation, La Jolla, 0.1% FBS. After 30 min, PMA or DMSO as CA) using 1 ml of the cDNA equivalent to 50 ng control, were added in the media for 10 min. mRNA with 1x Brilliant SYBR Green master Whole cell extracts were harvested in 1X mix and ROX reference dye (1:500) (Stratagene, lysis buffer [100mM tris(hydroxymethyl)ami-
Load more

Recommended publications
  • Anti-PRKCA / PKC-Alpha Antibody (Aa623-672) Rabbit Anti Human Polyclonal Antibody Catalog # ALS17942
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 Anti-PRKCA / PKC-Alpha Antibody (aa623-672) Rabbit Anti Human Polyclonal Antibody Catalog # ALS17942 Specification Anti-PRKCA / PKC-Alpha Antibody (aa623-672) - Product Information Application IHC-P, E Primary Accession P17252 Predicted Human, Mouse, Rat Host Rabbit Clonality Polyclonal Isotype IgG Calculated MW 76750 Anti-PRKCA / PKC-Alpha Antibody (aa623-672) - Additional Information Gene ID 5578 Alias Symbol PRKCA Other Names PRKCA, Aging-associated gene 6, AAG6, Alpha PKC, PKC-A, Pkcalpha, Protein kinase C alpha type, PKC Alpha, PKC-alpha, PKCA, Protein kinase c alpha, Protein kinase C, alpha Target/Specificity PRKCA Antibody detects endogenous levels of total PRKCA protein. Reconstitution & Storage Immunoaffinity purified Precautions Anti-PRKCA / PKC-Alpha Antibody (aa623-672) is for research use only and not for use in diagnostic or therapeutic procedures. Anti-PRKCA / PKC-Alpha Antibody (aa623-672) - Protein Information Name PRKCA Synonyms PKCA, PRKACA Function Page 1/4 10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 Calcium-activated, phospholipid- and diacylglycerol (DAG)- dependent serine/threonine-protein kinase that is involved in positive and negative regulation of cell proliferation, apoptosis, differentiation, migration and adhesion, tumorigenesis, cardiac hypertrophy, angiogenesis, platelet function and inflammation, by directly phosphorylating targets such as RAF1, BCL2, CSPG4, TNNT2/CTNT, or activating signaling cascade involving MAPK1/3 (ERK1/2) and RAP1GAP. Involved in cell proliferation and cell growth arrest by positive and negative regulation of the cell cycle. Can promote cell growth by phosphorylating and activating RAF1, which mediates the activation of the MAPK/ERK signaling cascade, and/or by up-regulating CDKN1A, which facilitates active cyclin-dependent kinase (CDK) complex formation in glioma cells.
    [Show full text]
  • Generation of the Catalytic Fragment of Protein Kinase C Alpha in Vasospastic Canine Basilar Artery
    Neurosurg Focus 3 (4):Article 4, 1997. Generation of the catalytic fragment of protein kinase C alpha in vasospastic canine basilar artery Motohiko Sato, M.D., Eiichi Tani, M.D., Tsuyoshi Matsumoto, M.D., Hirokazu Fujikawa, M.D., and Shinobu Imajoh-Ohmi, Ph.D. Department of Neurosurgery, Hyogo College of Medicine, Hyogo, Japan; and Institute of Medical Science, University of Tokyo, Tokyo, Japan In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated µ-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKC-alpha antibody shows a significant decrease in cytosolic 80-kD PKC-alpha and a concomitantly significant increase in membrane PKC-alpha in the spastic canine BA. In addition, an immunoblot study in which cleavage site­directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKC-alpha and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site­directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKC-alpha in spastic canine BA is translocated to the cell membrane, where PKC-alpha is rapidly cleaved into PKM as a result of proteolysis of the isozyme by µ-calpain but not by m-calpain.
    [Show full text]
  • PKC Alpha Rabbit Pab
    Leader in Biomolecular Solutions for Life Science PKC alpha Rabbit pAb Catalog No.: A13342 Basic Information Background Catalog No. Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that A13342 can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved Observed MW in diverse cellular signaling pathways. PKC family members also serve as major 85kDa receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The Calculated MW protein encoded by this gene is one of the PKC family members. This kinase has been 76kDa reported to play roles in many different cellular processes, such as cell adhesion, cell transformation, cell cycle checkpoint, and cell volume control. Knockout studies in mice Category suggest that this kinase may be a fundamental regulator of cardiac contractility and Ca(2+) handling in myocytes. Primary antibody Applications WB Cross-Reactivity Human, Mouse, Rat Recommended Dilutions Immunogen Information WB 1:500 - 1:2000 Gene ID Swiss Prot 5578 P17252 Immunogen Recombinant fusion protein containing a sequence corresponding to amino acids 523-672 of human PKC alpha (NP_002728.1). Synonyms PRKCA;AAG6;PKC-alpha;PKCA;PRKACA;PKC alpha Contact Product Information www.abclonal.com Source Isotype Purification Rabbit IgG Affinity purification Storage Store at -20℃. Avoid freeze / thaw cycles. Buffer: PBS with 0.02% sodium azide,50% glycerol,pH7.3. Validation Data Western blot analysis of extracts of various cell lines, using PKC alpha antibody (A13342) at 1:1000 dilution.
    [Show full text]
  • The Role of Z-Disc Proteins in Myopathy and Cardiomyopathy
    International Journal of Molecular Sciences Review The Role of Z-disc Proteins in Myopathy and Cardiomyopathy Kirsty Wadmore 1,†, Amar J. Azad 1,† and Katja Gehmlich 1,2,* 1 Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK; [email protected] (K.W.); [email protected] (A.J.A.) 2 Division of Cardiovascular Medicine, Radcliffe Department of Medicine and British Heart Foundation Centre of Research Excellence Oxford, University of Oxford, Oxford OX3 9DU, UK * Correspondence: [email protected]; Tel.: +44-121-414-8259 † These authors contributed equally. Abstract: The Z-disc acts as a protein-rich structure to tether thin filament in the contractile units, the sarcomeres, of striated muscle cells. Proteins found in the Z-disc are integral for maintaining the architecture of the sarcomere. They also enable it to function as a (bio-mechanical) signalling hub. Numerous proteins interact in the Z-disc to facilitate force transduction and intracellular signalling in both cardiac and skeletal muscle. This review will focus on six key Z-disc proteins: α-actinin 2, filamin C, myopalladin, myotilin, telethonin and Z-disc alternatively spliced PDZ-motif (ZASP), which have all been linked to myopathies and cardiomyopathies. We will summarise pathogenic variants identified in the six genes coding for these proteins and look at their involvement in myopathy and cardiomyopathy. Listing the Minor Allele Frequency (MAF) of these variants in the Genome Aggregation Database (GnomAD) version 3.1 will help to critically re-evaluate pathogenicity based on variant frequency in normal population cohorts.
    [Show full text]
  • AGC Kinases in Mtor Signaling, in Mike Hall and Fuyuhiko Tamanoi: the Enzymes, Vol
    Provided for non-commercial research and educational use only. Not for reproduction, distribution or commercial use. This chapter was originally published in the book, The Enzymes, Vol .27, published by Elsevier, and the attached copy is provided by Elsevier for the author's benefit and for the benefit of the author's institution, for non-commercial research and educational use including without limitation use in instruction at your institution, sending it to specific colleagues who know you, and providing a copy to your institution’s administrator. All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are prohibited. For exceptions, permission may be sought for such use through Elsevier's permissions site at: http://www.elsevier.com/locate/permissionusematerial From: ESTELA JACINTO, AGC Kinases in mTOR Signaling, In Mike Hall and Fuyuhiko Tamanoi: The Enzymes, Vol. 27, Burlington: Academic Press, 2010, pp.101-128. ISBN: 978-0-12-381539-2, © Copyright 2010 Elsevier Inc, Academic Press. Author's personal copy 7 AGC Kinases in mTOR Signaling ESTELA JACINTO Department of Physiology and Biophysics UMDNJ-Robert Wood Johnson Medical School, Piscataway New Jersey, USA I. Abstract The mammalian target of rapamycin (mTOR), a protein kinase with homology to lipid kinases, orchestrates cellular responses to growth and stress signals. Various extracellular and intracellular inputs to mTOR are known. mTOR processes these inputs as part of two mTOR protein com- plexes, mTORC1 or mTORC2. Surprisingly, despite the many cellular functions that are linked to mTOR, there are very few direct mTOR substrates identified to date.
    [Show full text]
  • N-Glycan Trimming in the ER and Calnexin/Calreticulin Cycle
    Neurotransmitter receptorsGABA and A postsynapticreceptor activation signal transmission Ligand-gated ion channel transport GABAGABA Areceptor receptor alpha-5 alpha-1/beta-1/gamma-2 subunit GABA A receptor alpha-2/beta-2/gamma-2GABA receptor alpha-4 subunit GABAGABA receptor A receptor beta-3 subunitalpha-6/beta-2/gamma-2 GABA-AGABA receptor; A receptor alpha-1/beta-2/gamma-2GABA receptoralpha-3/beta-2/gamma-2 alpha-3 subunit GABA-A GABAreceptor; receptor benzodiazepine alpha-6 subunit site GABA-AGABA-A receptor; receptor; GABA-A anion site channel (alpha1/beta2 interface) GABA-A receptor;GABA alpha-6/beta-3/gamma-2 receptor beta-2 subunit GABAGABA receptorGABA-A receptor alpha-2receptor; alpha-1 subunit agonist subunit GABA site Serotonin 3a (5-HT3a) receptor GABA receptorGABA-C rho-1 subunitreceptor GlycineSerotonin receptor subunit3 (5-HT3) alpha-1 receptor GABA receptor rho-2 subunit GlycineGlycine receptor receptor subunit subunit alpha-2 alpha-3 Ca2+ activated K+ channels Metabolism of ingested SeMet, Sec, MeSec into H2Se SmallIntermediateSmall conductance conductance conductance calcium-activated calcium-activated calcium-activated potassium potassium potassiumchannel channel protein channel protein 2 protein 1 4 Small conductance calcium-activatedCalcium-activated potassium potassium channel alpha/beta channel 1 protein 3 Calcium-activated potassiumHistamine channel subunit alpha-1 N-methyltransferase Neuraminidase Pyrimidine biosynthesis Nicotinamide N-methyltransferase Adenosylhomocysteinase PolymerasePolymeraseHistidine basic
    [Show full text]
  • PKC Alpha Affects Cell Cycle Progression and Proliferation in Human RPE Cells Through the Downregulation of P27kip1
    Molecular Vision 2009; 15:2683-2695 <http://www.molvis.org/molvis/v15/a285> © 2009 Molecular Vision Received 12 June 2009 | Accepted 3 December 2009 | Published 10 December 2009 PKC alpha affects cell cycle progression and proliferation in human RPE cells through the downregulation of p27kip1 Qianying Gao, Juan Tan, Ping Ma, Jian Ge, Yaqin Liu, Xuerong Sun, Lian Zhou State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China Purpose: Protein kinase C (PKC) plays an important role in the regulation of retinal pigment epithelium (RPE) cell proliferation. In this study, we investigated which of these isozymes could be responsible for the cell cycle and proliferation in human RPE cells. Methods: The effect of PKC activators on human RPE cell cycle progression was tested by flow cytometry. To identify the isoform of PKC responsible for the increased progression of the cells through the cell cycle, we monitored the effect of phorbol 12-myristate 13-acetate (PMA) on the subcellular localization of the nine PKC isoforms expressed in RPE cells. To evaluate the molecular mechanism by which PKCα induces cell cycle progression, we examined the transcript, protein, and cellular levels of cell cycle regulatory proteins using RT–PCR, western blotting, and a confocal microscope, respectively. Results: We demonstrated that PKC activation by PMA affected cell cycle progression in RPE cells. Of the nine PKC isoforms that were present in RPE cells, we found PKCα was both necessary and sufficient to promote cell cycle progression after being stimulated with PMA. Decreased PKCα expression resulted in a significant decrease in cell proliferation.
    [Show full text]
  • Distinct Regulation of Cardiac Fibroblast Proliferation and Transdifferentiation by Classical and Novel Protein Kinase C Isoform
    Molecular Pharmacology Fast Forward. Published on November 25, 2020 as DOI: 10.1124/molpharm.120.000094 This article has not been copyedited and formatted. The final version may differ from this version. Distinct regulation of cardiac fibroblast proliferation and transdifferentiation by classical and novel protein kinase C isoforms: possible implications for new antifibrotic therapies S. Tuuli Karhu, Heikki Ruskoaho, Virpi Talman* Affiliations Downloaded from STK, HR, and VT: Drug Research Program and Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Finland molpharm.aspetjournals.org *Corresponding author at ASPET Journals on September 30, 2021 1 Molecular Pharmacology Fast Forward. Published on November 25, 2020 as DOI: 10.1124/molpharm.120.000094 This article has not been copyedited and formatted. The final version may differ from this version. Running title: PKC agonists inhibit cardiac fibroblast activation Corresponding author: Virpi Talman Division of Pharmacology and Pharmacotherapy Faculty of Pharmacy University of Helsinki P.O. Box 56 (Viikinkaari 5E) FI-00014 Helsinki, FINLAND Downloaded from Tel: +358504480768 Email: [email protected] molpharm.aspetjournals.org Number of text pages: 35 Number of tables: 0 Number of figures: 4 at ASPET Journals on September 30, 2021 Number of references: 55 Number of words in the Abstract: 249 Number of words in the Introduction: 728 Number of words in the Discussion: 1459 Abbreviations: α-SMA, α-smooth muscle actin; aPKC, atypical protein kinase C isoform; BrdU, 5-bromo-2’-deoxyuridine; CF, cardiac fibroblast; cPKC, classical protein kinase C isoform; DDR2, discoidin domain receptor 2; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; HCA, high-content analysis; LDH, lactate dehydrogenase; MTT, 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nPKC, novel protein kinase C isoform; PKC, protein kinase C 2 Molecular Pharmacology Fast Forward.
    [Show full text]
  • Protein Kinase C As a Therapeutic Target in Non-Small Cell Lung Cancer
    International Journal of Molecular Sciences Review Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer Mohammad Mojtaba Sadeghi 1,2, Mohamed F. Salama 2,3,4 and Yusuf A. Hannun 1,2,3,* 1 Department of Biochemistry, Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA; [email protected] 2 Stony Brook Cancer Center, Stony Brook University Hospital, Stony Brook, NY 11794, USA; [email protected] 3 Department of Medicine, Stony Brook University, Stony Brook, NY 11794, USA 4 Department of Biochemistry, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Dakahlia Governorate, Egypt * Correspondence: [email protected] Abstract: Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. Citation: Sadeghi, M.M.; Salama, PKC isoforms α, ", η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates M.F.; Hannun, Y.A. Protein Kinase C with worse prognosis in NSCLC patients.
    [Show full text]
  • Effectors and Potential Targets Selectively Upregulated in Human KRAS-Mutant Lung Adenocarcinomas
    bioRxiv preprint doi: https://doi.org/10.1101/041137; this version posted June 4, 2016. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Effectors and potential targets selectively upregulated in human KRAS-mutant lung adenocarcinomas Jinyu Li1, Raffaella Sordella2, and Scott Powers1,2,3 1Department of Pathology, Stony Brook University, Stony Brook, NY, 11794 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724 3Corresponding author, email [email protected] Abstract Genetic and proteomic analysis of human tumor samples can provide an important compliment to information obtained from model systems. Here we examined protein and gene expression from the Cancer Genome and Proteome Atlases (TCGA and TCPA) to characterize proteins and protein-coding genes that are selectively upregulated in KRAS-mutant lung adenocarcinomas. Phosphoprotein activation of several MAPK signaling components was considerably stronger in KRAS-mutants than any other group of tumors, even those with activating mutations in receptor tyrosine kinases (RTKs) and BRAF. Co-occurring mutations in KRAS-mutants were associated with differential activation of PDK1 and PKC-alpha. Genes showing strong activation in RNA-seq data included negative regulators of RTK/RAF/MAPK signaling along with potential oncogenic effectors including activators of Rac and Rho proteins and the receptor protein-tyrosine phosphatase genes PTPRM and PTPRE. These results corroborate RAF/MAPK signaling as an important therapeutic target in KRAS-mutant lung adenocarcinomas and pinpoint new potential targets. Introduction How mutationally activated KRAS and other canonical RAS genes malignantly transform cells and how to block this process for therapeutic benefit has been a subject of intense investigation for over thirty years.
    [Show full text]
  • Characterization of the Small Molecule Kinase Inhibitor SU11248 (Sunitinib/ SUTENT in Vitro and in Vivo
    TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Genetik Characterization of the Small Molecule Kinase Inhibitor SU11248 (Sunitinib/ SUTENT in vitro and in vivo - Towards Response Prediction in Cancer Therapy with Kinase Inhibitors Michaela Bairlein Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ. -Prof. Dr. K. Schneitz Prüfer der Dissertation: 1. Univ.-Prof. Dr. A. Gierl 2. Hon.-Prof. Dr. h.c. A. Ullrich (Eberhard-Karls-Universität Tübingen) 3. Univ.-Prof. A. Schnieke, Ph.D. Die Dissertation wurde am 07.01.2010 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 19.04.2010 angenommen. FOR MY PARENTS 1 Contents 2 Summary ................................................................................................................................................................... 5 3 Zusammenfassung .................................................................................................................................................... 6 4 Introduction .............................................................................................................................................................. 8 4.1 Cancer ..............................................................................................................................................................
    [Show full text]
  • Study of Z-Disc-Associated Signaling Networks in Skeletal Muscle Cells by Functional and Global Phosphoproteomics
    PHDTHESIS Study of Z-disc-associated Signaling Networks in Skeletal Muscle Cells by Functional and Global Phosphoproteomics Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Lena Reimann geboren in Bielefeld Freiburg im Breisgau 01.08.2016 Angefertigt am Institut für Biologie II AG Biochemie und Funktionelle Proteomforschung zellulärer Systeme unter der Leitung von Prof. Dr. Bettina Warscheid Dekan der Fakultät für Biologie: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Stefan Rotter Betreuer der Arbeit: Prof. Dr. Bettina Warscheid Referent: Prof. Dr. Bettina Warscheid Koreferent:Prof. Dr. Jörn Dengjel Drittprüfer: Prof. Dr. Gerald Radziwill Datum der mündlichen Prüfung:21.10.2016 ART IS I, science is we. - Claude Bernard Zusammenfassung Als essentielle, strukturgebende Komponente des Sarkomers spielt die Z-Scheibe eine maßge- liche Rolle für die Funktionalität der quergestreiften Muskulatur. Die stetige Identifizierung von neuen Z-Scheiben-lokalisierten Proteinen, sowie deren Relevanz in muskulären Krankheits- bildern, hat die Z-Scheibe zunehmend in den Fokus der aktuellen Forschung gerückt. Neben ihrer strukturgebenden Funktion zeigen neuere Studien, dass die Z-Scheibe ein Hotspot für Signalprozesse in Muskelzellen ist. Bisher gibt es jedoch keine globalen Untersuchungen zur Aufklärung der komplexen Signalwege assoziiert mit dieser Struktur. Um Z-Scheiben-assoziierte Signalprozesse näher zu charakterisieren, wurde im ersten Teil dieser Arbeit eine großangelegte Phosphoproteomstudie mit ausdifferenzierten, kon- trahierenden C2C12 Myotuben durchgeführt. Zu diesem Zweck wurden die tryptisch ver- dauten Proteine mittels SCX-Chromatographie fraktioniert. Die anschließende Phosphopep- tidanreicherung erfolgte mit Titandioxid, gefolgt von einer hochauflösenden massenspek- trometrischen Analyse. Insgesamt wurden 11.369 Phosphorylierungsstellen, darunter 586 in sarkomerischen Proteinen gefunden.
    [Show full text]