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  192. SSeCKS maskiert Cyclin D1 in glomerul盲ren parietalen Epithelzellen und Einfl眉sse proliferative Verletzung in der glomerulusBettina Burnworth1, Jeff Pippin1, Prasanthi Karna1, Shin Akakura2, Ron Krofft1, Guoqiang Zhang1, Kelly Hudkins3, Charles E Alpers1, 3, Kelly Smith3, Stuart J Shankland1, Irwin H Gelman2 und Peter J Nelson11Division f眉r Nephrologie und Kidney Research Institute, University of Washington, Health Sciences Center, Seattle, WA, USA2Roswell Park Cancer Institute, Buffalo, NY, USA3Department f眉r Pathologie der Universit盲t von Washington, Seattle, WA, USACorrespondence: Dr. PJ Nelson, MD, Abteilung f眉r Nephrologie und Kidney Research Institute, University of Washington, Health Sciences Center, Zimmer BB1259, 1959 NE Pacific Street, Seattle, WA 98195, USA. Hier zeigen wir, dass die Src unterdr眉ckt Proteinkinase C Substrat (SSeCKS), ein multivalentes Ger眉st A-Kinase-Ankerprotein, sondert Cyclin D1 in das Zytoplasma der Ruhe BBW. SSeCKS Expression in embryonalen induzierten PECs, aber nicht in embryonalen podocytes, beginnend an der S-Phase des glomerulogenesis und konstitutiv postnatal nach PECs ausgedr眉ckt, aber nicht podocytes im normalen Glomeruli. Cyclin D1 wurde mit SSeCKS aus gekapselt Glomeruli enthalten BBW immunpr盲zipitiert, w盲hrend entkapselten Glomeruli ohne BBW fehlte SSeCKS und Cyclin D1. Zellkontakt Inhibierung der Proliferation an kultivierten induzierten PECs SSeCKS Expression und die Bindung von Cyclin D1 durch SSeCKS im Zytoplasma, w盲hrend SSeCKS Phosphorylierung von Proteinkinase C Bindung gest枚rt, was zu der nukle盲ren Translokation von Cyclin D1.As Vorstufen f眉r podocytes in postnatalen Glomeruli die unterschiedlichen inneren Mechanismen, die f眉r die wiederholte Perioden der Zellzyklusarrest erm枚glichen und neu Eintrag von BBW abgesehen von Podozyten sind unknown.6 Gegensatz der proliferativen Unabh盲ngigkeit von embryonalen Stammzellen von G1 nach S Checkpoint Kontrolle durch Retinoblastom-Protein (Rb), Forschung auf ruhenden Vorfahren in anderen postnatale Gewebe legt nahe, dass mitogeninduzierte Cyclin D1 Cyclin-abh盲ngigen Kinase 4 Phosphorylierung von Rb treibt ihre Zellzyklus progression.6 Interessant ist, in den letzten Stadien der glomerulogenesis, wuchernden embryonalen Podozyten herunterregulieren Cyclin D1 und Cyclin D3 hochregulieren und die G1-S Zellzyklus Hemmer, p27 und p57, Ausdruck, der konstitutiv in reifen Podozyten in der postnatalen glomeruli.7 bleibt, 8 Dieses Terminal Zellzyklus-Zustand f眉r differenzierten Podozyten ist nicht ersichtlich, f眉r BBW, im Einklang mit dessen Funktion als zusammenh盲ngende progenitors.1, 2 jedoch der Zelle Zyklus Mechanismen, die deutlich den Ruhezustand und Proliferationspotential von BBW pflegen sind unknown.Here, wir vermuten, dass Src unterdr眉ckt Proteinkinase C Substrat (SSeCKS), 9 von der Nager Ortholog des menschlichen gravin codiert maskiert Cyclin D1 in Maus-BBW, als eine Portion Gating-Pr眉fpunkt f眉r Zellzyklusarrest. SSeCKS ist ein zytoplasmatisches, multivalente Ger眉stprotein in der Familie von A-Kinase-Ankerproteine 鈥嬧€?AKAPs) 10, die urspr眉nglich als Tumor suppressor.11, 12 SSeCKS ist einzigartig unter AKAPs durch mit Proteinkinase C (PKC)-Phosphorylierungsstellen, die Cyclin flankieren kloniert wurde Bindungsmotiven, die Cyclin D1.13, 14 verbinden, in Reaktion auf Zellkontakt Proliferationshemmung wird 15 SSeCKS Expression induziert, was zur Bindung von Cyclin D1 bestehende Protein im Zytoplasma und die Herunterregulierung von weiteren Cyclin D1-Expression durch extrazellul盲res Signal geregelt Kinase-abh盲ngige Signalwege. Dies f眉hrt zu Wachstumsstillstand durch Hemmung G1 zu S Zellzyklus progression.13, 16 Phosphorylierung von SSeCKS durch aktivierte PKC (aPKC) sind Zeichen von vielen Mitogene, 17 gibt diese Bindung von Cyclin D1, so dass seine nukle盲re Translokation und G1 zu S-Zelle induziert Zyklus progression.13, 14 In fr眉heren Studien haben wir entdeckt, dass SSeCKS wird von reifen BBW ausgedr眉ckt, ist aber nicht in reifen Podozyten nachgewiesen, in der postnatalen glomeruli.18 Diese differentielle Expression erh枚ht die M枚glichkeit, dass SSeCKS ist bei der Aufrechterhaltung der Ruhezustand deutlich beteiligten und Proliferationspotential von BBW abgesehen von Podozyten durch die Regelung von Cyclin D1.Top pageMATERIALS UND METHODSMiceAll Experimenten an M盲usen wurden durch Institutional Animal Care und Verwenden Committee genehmigt. Magnetic Bead Isolierung gekapselt und decapsulated Glomeruli aus Wildtyp-M盲usen nach der Differenzgr枚脽e Siebung des Faul Nieren wurde wie zuvor beschrieben durchgef眉hrt, 23 mit isolierten Glomeruli sofort in RIPA-Puffer (Teknova, Hollister, CA, USA), die komplette Protease-Inhibitor (Roche lysiert Indianapolis, IN, USA), 50 Natriumfluorid und 0,1 Natriumorthovanadat bei 4 zum Gesamtprotein zu sammeln. Urin wurde von jeder Maus an Tag 14 nach der zweiten intraperitoneale Injektion unmittelbar vor der Erhebung, Formalin-Fixierung und Paraffineinbettung von Nieren gesammelt. Urin wurde f眉r die Konzentration von Albumin und Kreatinin mit der Albuwell Murine Mikroalbumin ELISA (Exocell, Philadelphia, PA, USA) und Kreatinin Farbmetrische Mikroplatten-Assay (Cayman Chemical, Ann Arbor, MI, USA), die jeweils nach den Protokollen des Herstellers untersucht. ImmunofluorescenceImmunofluorescence Erkennung von SSeCKS, phospho SSeCKS ser283, und Cyclin D1 in kultivierten BBW wurde wie beschrieben previously.18 Immunfluoreszenz-Nachweis von SSeCKS und Cyclin D1 in 4 Abschnitte Nieren durchgef眉hrt wurde durchgef眉hrt, nachdem Abschnitte wurden von Paraffin (Histoclear gel枚scht; National Diagnostics, Atlanta, GA , USA), rehydratisiert Antigen in 10-Citrat (pH 6,0), abgerufen durch Mikrowellen Siedepunkt und 5 mit Rinderserumalbumin blockiert. Prim盲re Antik枚rper wurden folgende: Kaninchen polyklonalen Anti SSeCKS (Irwin Gelman Laboratory); polyklonalen Kaninchen anti phospho SSeCKS ser283 (Irwin Gelman Laboratory) und monoklonalen anti Cyclin D1 (Millipore, Temecula, CA, USA). Sekund盲re Antik枚rper waren die folgenden: Alexa 488-konjugierten Anti-Kaninchen-IgG (Invitrogen, San Diego, CA, USA) und TXR konjugierten Anti-Maus-IgG (Invitrogen). Die Zellkerne wurden mit Hoechst 33342 (Thermo Scientific, Natick, MA, USA) gef盲rbt. Warp Chromogen Red Kit (Biocare Medical, Concord, CA, USA) wurde auch f眉r Sekund盲r Nachweis von Cyclin D1 mit wei脽em Licht und Fluoreszenz in Nierenschnitten ohne zus盲tzliche Flecken verwendet. Imaging wurde auf einem DMRB Fluoreszenz-Mikroskop (Leica, Richmond, IL, USA) durchgef眉hrt. West BlottingProtein Lysate wo durch SDS-PAGE getrennt und auf PVDF-Membranen (Millipore) f眉r Immunoblotting 眉berf眉hrt. Und PAX8 (1:500 Gesamtprotein aus isolierten Glomeruli wurde SSeCKS (1:200), Cyclin D1 (1:500), Cyclin D3 (;; monoklonalen Maus-Lab Vision, Fremont, CA, USA 1:500) immungeblottet; polyklonalen Kaninchen; Proteintech Group, Chicago, IL, USA), mit Tubulin (1:500; monoklonalen Maus-Maus; Lab Vision) als Ladekontrolle. Gesamtprotein aus differenzierten BBW mit PKC aktiviert oder gehemmt wurde f眉r phospho SSeCKS ser283 (1:200) immungeblottet mit SSeCKS als seine eigene Lade Referenz. F眉r die Immunpr盲zipitation von Cyclin D1 mit SSeCKS, 100 Protein aus verkapselten Glomeruli oder aus kultivierten differenzierten PECs wurde mit polyklonalen Kaninchen-anti SSeCKS Antik枚rper 眉ber Nacht bei 4 gefolgt von der Bindung an Protein A-Agarose-PLUS (Santa Cruz Biotechnology, Santa Cruz, CA, USA inkubiert ) f眉r die SDS-PAGE-Trennung von Cyclin D1 und SSeCKS. Der umgekehrte Immunpr盲zipitation von SSeCKS mit Cyclin D1 als K枚der wurde auch kultiviert PECS durchgef眉hrt. Prim盲r-Antik枚rper wurden unter Verwendung von HRP-konjugiertem Antik枚rper aktiviert Chemilumineszenz (Amersham, Piscataway, NJ, USA) auf einem ChemiDoc EQ System (BioRad Laboratories, Berkeley, CA, USA) nachgewiesen abgebildet. Gezeigt Western-Blots sind repr盲sentativ f眉r drei separate experiments.Quantitative HistopathologyThe Ausma脽 der zellul盲ren Ver盲nderungen in den Glomeruli jeder Maus mit NTN wurde von Nieren Pathologen von der Universit盲t von Washington Pathology Laboratory Research Service naiv zu Versuchsprotokoll quantifiziert. Periods盲ure-Schiff gef盲rbt Nieren koronalen Schnitte wurden verwendet, um die Prozent der gesamten Glomeruli mit jedem Mobilfunk L盲sion zu berechnen. Nach der Herstellung von 4 Nierenschnitten, wie oben beschrieben, die immunhistochemische F盲rbung von PAX8, Cyclin D1 oder Wilms-Tumor-Antigen (WT1, Santa Cruz Biotechnology) in den Nieren war durch sekund盲re Detektion mit biotinylierten Antik枚rpern und ABC Elite (Vector Laboratories, Burlingame, CA , USA)-System, gem盲脽 dem Protokoll des Herstellers. Die Anzahl der PAX8 positiven Kernen oder Cyclin D1 positive Keime pro erkrankten Glomerulus in jeder Maus mit NTN wurde durch Dividieren der Anzahl der positiven gef盲rbten Kerne in allen Glomeruli mit zellul盲ren Verletzungen durch die Anzahl der Glomeruli mit zellul盲ren Verletzungen in koronalen Nieren section.StatisticsAll Werte berechnet ausgedr眉ckt als mittlere statistische Signifikanz wurde durch die zweiseitigen t-Test gepr眉ft. Die Signifikanz wurde auf dem Niveau 0,05 von probability.Top von pageRESULTSSSeCKS und Cyclin-D1 in P茅cs, w盲hrend und nach GlomerulogenesisDuring glomerulogenesis embryonale Podozyten aber nicht embryonalen BBW konstitutiv p27 und p57 ab der Kapillare Schleifenstufe und Weiterbildung bis in die postnatale Leben, Kennzeichnung testeten ihre Ausgangszellzyklus als terminal differenziert Bestandteile der glomerul盲ren Filtrationsbarriere (GFB) .8, das Entwicklungsprogramm, das separat stellt die Ruhe proliferative Potenzial von PEC als Vorl盲ufer in der postnatalen Nieren ist jedoch unbekannt. So untersuchten wir die Expression von SSeCKS durch Immunfluoreszenz (IF) in pre natal Maus Nieren bei E18, die alle Stufen der glomerulogenesis aufweist. SSeCKS nicht in embryonalen Nierenzellen bildet Bl盲schen, ureteric Knospen, oder Komma Formk枚rper aus Ur Glomeruli erkannt (Abbildung 1a und b). Intarsien in der unteren rechten Ecke des Panels umrei脽en die durch Fluoreszenzmikroskopie gezeigt, Anatomie, in der Kerne Fleck blau, SSeCKS Flecken Gr眉n und Cyclin D1 Flecken rot. (A) SSeCKS nicht in embryonalen Nierenzellen bilden Bl盲schen oder Harnleiter Knospen festgestellt. (B) SSeCKS nicht in embryonalen Zellen bilden Komma Formk枚rper nachgewiesen. (C) SSeCKS wird von embryonalen parietalen Epithelzellen (BBW), die die Oberfl盲che des parietalen S-f枚rmigen K枚rper ausgedr眉ckt. (D) Cyclin D1 in den Kernen der SSeCKS negativen embryonalen podocytes Bildung der Eingeweideoberfl盲che S f枚rmigen K枚rper (Pfeile), angrenzend an SSeCKS positiven embryonalen PECs ausgedr眉ckt. (E) SSeCKS wird von embryonalen BBW an der Kapillare Schleifenstufe ausgedr眉ckt. (F) Cyclin D1 ist nicht im negativen SSeCKS embryonalen Podozyten oder in SSeCKS positiven embryonalen BBW an der Kapillare Schleife Stadium erkannt. (G) SSeCKS wird von embryonalen BBW in voll ausgebildet Glomeruli ausgedr眉ckt. (H) Die Expression von SSeCKS an PEC durch den 脺bergangszonen mit angrenzenden Epithel-Zellen an der Gef盲脽 (Pfeile) und Harnwege (Pfeilspitzen) Pole der Bowman-Basalmembran (BBM) abgegrenzt begrenzt. (I) Western Immunoblots von Protein-Lysaten aus gekapselt und decapsulated Glomeruli aus separaten M盲usen zeigen, dass SSeCKS und Cyclin D1 sind nicht reichlich in decapsulated Glomeruli fehlt paired box gene 8 (PAX8) positive BBW getrennt, jedoch unter Beibehaltung Cyclin D3 positive Podozyten. (J) Western Immunoblots (IB), der die Bindung von Cyclin D1 bis SSeCKS immunpr盲zipitiert (IP) von Protein-Lysaten von gekapselten Glomeruli aus separaten M盲usen isoliert. Vergr枚脽erungen: 400 Tafeln in a, b, c, e, f, g und h; 2000 Tafel d.Full Figur und Legende (523K) Dieser deutliche Expression von SSeCKS am S Stadium der embryonalen BBW, aber nicht in der embryonalen Podozyten , war auch an der Kapillare Schleifenstufe (Abbildung 1e) und in P茅cs voll ausgebildet Glomeruli (Abbildung 1 g) vorhanden, letztere zuvor in postnatalen kidneys.18 gezeigt Das begrenzt Ausdruck SSeCKS von reifen BBW war an den 脺bergangszonen zwischen BBW deutlich und anderen angrenzenden Epithelzellen an den Harn-und Gef盲脽 Pole BBM (Abbildung 1h). Wir best盲tigten diese suggestive Erkenntnisse aus IF durch Immunpr盲zipitation Cyclin D1 mit SSeCKS aus isolierten Glomeruli. Nur gekapselt Glomeruli enthalten, BBW, durch Positivit盲t f眉r paired box gene 8 (PAX8) gekennzeichnet, 25 ausgedr眉ckt reichlich SSeCKS und Cyclin D1 (1i). Im Vergleich decapsulated Glomeruli fehlt BBW, aber Beibehaltung Cyclin D3 positive Podozyten, keine signifikanten Cyclin D1 oder SSeCKS auszudr眉cken. Diese differentiell exprimiert in gekapselten Glomeruli mit SSeCKS (Abbildung 1 j) immunpr盲zipitiert Cyclin D1 vorhanden. Zusammengenommen deuten diese Ergebnisse, dass SSeCKS kann bei der Aufrechterhaltung der Ruhezustand des BBW abgesehen von Podozyten durch Bindung und eine stabile Sequestrierung Pool von Cyclin D1 deutlich beteiligt werden, die Mechanismen, die w盲hrend glomerulogenesis.SSeCKS und Cyclin-D1 in kultivierten PECsTo initiieren weiter pr眉fen die Steuer von Cyclin D1 durch SSeCKS in P茅cs, untersuchten wir die Expression und bindende Wechselwirkung von Cyclin D1 und SSeCKS in kultivierten PECs.23 Erh枚hung Zellkontakt zwischen BBW von 50 bis 100 Zellkonfluenz SSeCKS induzierte Expression im Zytoplasma, gleichzeitig mit der Umverteilung von Cyclin D1 aus Kernf盲chern und den zytoplasmatischen markiert Herunterregulation der Gesamt Cyclin D1-Expression (Abbildung 2a und b), im Einklang mit fr眉heren data.13, 14, 15, 16 Diese zytoplasmatischen Cyclin D1 immunpr盲zipitiert mit SSeCKS (Abb. 2b und Erg盲nzungs Abbildung 2), auch nach Differenzierung vollst盲ndig konfluenten BBW 眉ber 14 Tage, um den Zustand der reifen BBW im normalen Glomeruli, 23 mauern unsere Ergebnisse dieser Bindungswechselwirkung zwischen SSeCKS und Cyclin D1 in einem stabilen Zustand in vivo nachahmen. (A) Fluoreszenz-Detektion SSeCKS (gr眉n), Cyclin D1 (rot) und Kerne (blau) bei zunehmender Zellkontakt zwischen PECs von 50 bis 100 Zellen Konfluenz, der die Induktion von SSeCKS im Zytoplasma mit begleitenden Kern cytoplasmatische Verteilung von Cyclin D1 , Kompartimentierung, die in vollst盲ndig differenzierten (FD) BBW bleibt. (B) Western Immunoblots (IB), die Herabregulation von Cyclin D1 und Immunpr盲zipitation (IP) mit zytoplasmatischen SSeCKS bei zunehmender Zellkontakt zwischen BBW von 50 bis 100 Zellkonfluenz, Bindung, bleibt in ausdifferenzierten PECs.Full Figur und Legende (264K) SSeCKS ein Substrat f眉r aPKC, 12 und Phosphorylierung von SSeCKS aPKC gibt seine Ger眉stfunktion, wodurch cytoplasmatische nukleare Translokation von Cyclin D1.13, 14 unter Verwendung eines monoklonalen Antik枚rpers, der spezifisch erkennt die Phosphorylierung von Serin 283 in SSeCKS, eine Zielrest f眉r aPKC, 12 haben wir festgestellt schnelle Phosphorylierung von SSeCKS in P茅cs nach Aktivierung von PKC durch Phorbolester, Phorbol 12,13-diacetat, oder durch l枚sliche TNF letztere eine bekannte PKC activator26 und Entz眉ndungs 鈥嬧€婱itogen in den proliferativen podocytopathies (Abbildung 3) .27, 28 Diese Phosphorylierung wurde von der pan PKC-Inhibitor Staurosporin-Aglycon aufgehoben. Der unmittelbare fr眉he Hochregulation von SSeCKS Ausdruck, der mit mitogenen Signal tritt sich auch in PECs.11, 16 Als separate Steuer zeigte Maus-Fibroblasten exprimieren SSeCKS auch Phosphorylierung an Serin 283 nach Aktivierung von PKC (Bild 3b). Cyclin D1 verteilt aus dem Zytoplasma in den Zellkern in PECs mit Phosphorylierung durch SSeCKS aPKC (Abbildung 3c). Zusammengenommen deuten diese Ergebnisse, dass SSeCKS stabil maskiert Cyclin D1 im Zytoplasma von BBW bei Wachstumsstillstand in Zellkontakt, mit Verlust dieser Abschottung der Cyclin D1 folgenden Phosphorylierung von SSeCKS von aPKC. (A) Western Immunoblots zeigt schnelle Phosphorylierung an Serin 283 SSeCKS (phospho SSeCKS ser283) innerhalb von 5 der Aktivierung von PKC in P茅cs von Phorbol 12,13-diacetat (PDA) oder Tumor-Nekrose-Faktor (TNF Signalisierung durch Staurosporin-Aglycon (Inhibitor aufgehoben). (b) Steuer westlichen Immunoblots, die die gleiche wie in Signaltafel in eine Maus-Fibroblasten exprimieren SSeCKS. (c) Fluoreszenz-Detektion von phospho SSeCKS ser283 (gr眉n), Cyclin D1 (rot) und Zellkerne (blau) in vollst盲ndig differenzierten BBW zeigt Phosphorylierung von SSeCKs und Cytoplasma Kern Umverteilung von Cyclin D1 nach Aktivierung von PKC. SSeCKS, unterdr眉ckt Src-Proteinkinase C Substrat.

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