Borasio, Gian Domenico

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Differential effects of the protein kinase inhibitor K-252a on the in vitro survival of chick embryonie neurons Gian Domenico Borasio Max-Plal1ck-lnstitute for PsydriUlry, O ... parlmenl of N ... u,oc}wmü/ry. Murlin.fried ( F_RG) (Receiv~d t I August 1989: Revised version received 28 August 1989; Accepled 28 August 1989) Kl'y.wrds: K-252a; Protein kinase inhibitor: Nerve growlh faclor; eiliary neuronotrophk factor; Embryonic neuron; Signaltransduclion Thc cffects or the protein kinasc inhibitor K-252a on the in vitro survival or different populations of chick embryonic neurons wcrc teste<!. Followingdissociation from thecorrcsponding ganglia by tri lur .. tion, thc neurons wert: cultured on laminin-coatcd dishcs in both tbc presencc and absence of thcir rcspeclive neurotrophic faclor. K-252a alune promoted long-term neuronal survival ofdorsal TOot gang lion IORG) and ciliary ganglion (CG) neurons in a dose-depcndent fashion . No soch effect was seen with sympathetic ganglion (SG) neurons. In addition. K-252a did nOI interferc wilh nerve growth ractor (NGFr or ciliary ncuronot rophic factor (CNTF)-mediated survival of DRG or CO neurons. rcspeclively. but comple lcly blocked thc NGF· and CNTF-induced survival of SO neurons. High potassium-induced survival or SG neurons was not affectcd by K-252a. These rcsults point to differenccs bctween various neuronal popula-tions in the signal transduction mcchanism for neurotrophic factors. h is now widely recognized that the survival of vertebrate embryonic neurons depends on target-derived neurotrophic factors, nerve growth factor (NGF) being so far the most st udied molecule [3.19]. Despite extensive studies, stil1little is known about the signal transduction of neurotrophic factors, including NGF, resuhing in neuronal survival and/or fiber oUlgrowth. Recent results obtained in PC I2 cells [4,10,18] and cult ured neurons [5] support the concept of a central role for the ra.f proto-oncogene protein in the signalling cascade initiated by the binding of neurotro-phic factors to their specific receplors. However, few data are available regarding the signalling steps upstream and downstream ofthe ras protein. One ofthe major difficuhies for such studies has been the lack ofagents specifically blocking the actions of neurotrophic factors. Recently, a new protein kinase inhibitor o f microbial origin, K-252a [14J, has been found to inhibit specifically a number of NGF-induced responses in the NGF-sensitive pheochromocytoma cell line PC I2 Correspondl'nl'l': G.O. Borasio. Presen! addrcss: Neurological Clinic. University of Munieh. Klinikum Grosshadem. Marchioninistrdssc 15. D-8000 München 70. F.R.G. [11.15]. In addition. K-252a has bcen reported to inhibit hoth NGF-i ndueed neurite outgrowth in cultured chick embryo dorsal rOOI ganglion (DRGl cells [17]. and NGF-dependent increase in neurofilament protein expression in eultured human embryonie DRG neurons [7]. In order 10 further examine the effects 0 1' K-2 52a. I lesled this suhsta ncc on 3 differeIH neuronal populations iso la ted from the ehick em-hryo. Chiek dorsal root ga nglion (D RG) neurons at em bryonic day 9 (E9). cil iary gang-lion (CG) neurons at E8 and sympat het ic ganglion (SO) ne urons al E 12 were isolated from the eorrespondin g gangli a und cullurcd using previo usly described methods [5.8]. After preparatio n. the gangl ia were trypsi nizcd (0.2% trypsin in PBS for 30 min ) and di ssociatcd by mild tritura tion. The cell suspension was prepla ted as dcscribcd fl6] on Nune lissue eulture dishes. The neuron-enriehed cell suspensions were plated on Costar 24-well plates ( 1.6 cm weil diameter) whieh had been eoated seq uenlially with poIY-DL-ornithine (0.5 mg/mi. Sigma) and laminin (5 Jlgjml. BRL) as deseribed [6.9]. The cell densilY was 10.000 cells/well (8.000 fo r ciliary neurons). Non-neu ronal cells eom prised < 5% of the (Olal populalion. Immediately after plating. fac(Ors werc added 10 the appropriatc wells at thc following final concenlrations: NGF 20 ng/ml. CNTF 10 ng/ml. K 1 35 mM. K-252a 50-800 nM. NGF and CNTF were purified as described [12.13]. The eu ltures were mainta incd wilh F I4 medium (Gibeo) con-taining 10% heal-inactivaled horse serum (Boeh ringer Mannheim) at 37"C and 3.5% CO: in a humidified environment. 24 to 48 h after plating. approx. 1/801' the weH surface was scanned fo r Ihe presence of phase-bright cells_ Thi s procedure was repeatcd afte r 8 and 15 days to establi sh long-Ierm survival rates. Cell counting was perfo rmcd on a Leitz i nv~ned microscope. magnification 125 x . On E9 DRG neurons. <I dose-dependelll surviva l effect of K-252a ahme was obscrvcd (Fig. IA). The highest survival rate was found at a eoncentration of 800 nM and was virtually idenlical wilh the survivallevels rout inely obtained wit h NGF in th ls system. The NGF-induced su rvival was marginall y impaired al K-252a con-ccntrations ~200 nM. Morphologically. howevcr.lhc DRG neurons cu ltured in Iht! presence of K-252a showed kss pronou nced cel l body hypen rophy as compa red to Ihc NGF·t reated o nes (Fig. 2A Cl. In addilion. neurite outgrowth induced by K-252a resulted in finer and more branched proccsses (Fig. 2 B). Thc survival effect induced by K-252a persistt!d wilhout sign ifican t decrease fo r over two wecks (data nOI shown). Simila r results were obtained wi th E8 ciliary ganglion neurons (Fig. I B). Agai n. K-252a promotcd neuronal surviva l in a dose-dependent fashion. and did not illler-fe re wit h CNTF-induced survival. Mo rph ologica l changes were comparable 10 those observed in DRG neurons (Fig. 2D- F). Again. neurite outgrowlh and arbo rizat ion were enhanced and the processes appcared thinner than in the control cult ures. Surprisingly. a completely different picture was observed using E I2 sym pa thel ic neurons. Here. K·252a had no intrinsic surviv<l l-promolin g activity. but inhibited vir-tual1y eompletely thc survival effecls of NGF. The sa me result was observed with CNTF. which is also known to promote the in vitro survival ofsy mpathclic neurons [2]. That thc inhibilory ,IClion of K-252a was not due to a genera l loxicity of thi s A -• • • " .. 00 ,, B - . . -.--.-----K-252a [n t.l ] K- Z528 [n tol l c .c: Il000 /0_ 0-----"-_0 • :,; ::f\:~. ~ \~ E l2 SG ~ ,~ "---.--------------u ~ e • • '00 '00 .00 K-2520 [nM) Fig. ( Elfects of K-152a on cullUred chick embryonic neurons. CelJs weil: cultured as dc:st:ribed in the tC)(! and grown in thc presencc of the indica ted K-152a conce l1trations only (filIed c ircles) or together with 10 ngjml NGF (emply sqU3Te$), 10 ngjml CNTF (cmply triangles), or 35 mM K ' (empty circles) fo r 48 h (24 h for ciliary neurons). A: E9 D RG neurons. B: ES ciliary neurons. C: EI l sympathet ic neurons. suhslance was demonstrated by the raC] thai survival induced by high (35 mM ) K "' concentration was not affected (Figs. Je and 3). The data obtained with K-252a on sympathetic neurons are reminiscent of Ihe ef-reets of the methyltransferase inhibitor 5'-deoxy-5'-methylthioadenosine (MTA) on Ihe same ceHs. Like K-252a, MTA at 3 mM was shown 10 specifically inhibit NGF-. bul not high K +--induced surviva l of EI 2 sympalhetic neurons [IJ. The observation ofthe inlnnsic K-252a survival activity on DRG and ciliary neu-rons was somewhal unexpected for two reasons: indeed , previo us reports had de-scribed inhibito ry effects of the action o f NGF on DRG neurons by K-252a [7. 17]. In addi tion , K-252a at concentratio ns > 200 nM is known to be toxic, at least on pe l2 reHs [1 5). However, in previo us reports dealing with the effects of K-252a on cuhured neurons, no quantitative survi val assessmenl was performed. and no data were shown regarding the effects o f K-252a alone at concentrations > 50 oM . At present, it is difticult to offer a satisfactory explanation for the discrepancy belween the observations reported here and some previous reports [7,17]. The remarkable discrepancy between the elfects of high concentrations of K-252a o n either cilia ry and sensory neurons on Ihe one hand (promotion of cell survi val) and on sympathetic neurons on the o ther (bl ockade of NG F- or CNTF-induced survival) l'W'_, ., .~ r ~~. '.! "-', . ~.'.. .1 • ~:J~ Fig. 2. Pbase micrographs of E9 DRG (A-C) and E8 ciliary (D- F) neurons cultured for 48 b (24 h for ciliary neurons) in thc presence of: (A) NGF (20 ng/mI). (B.E) K-252a (800 nM ), (C) NGF + K-252a. (0 ) CNTF 10 nglml. CF) CNTF + K-252a. Note thc smaller ccll body size in Band C as comparcd 10 A and thc pronounced neurite outgrowth in B. E and F. Magnilkat ion 290 x. Fig. 3. Phase micrographs ofEI2 sympathctic neurons cultured for 48 h in the absence (A.e,E) or presence (B,O.F) or 800 nM K· 252a. Factors added to the cultures wcre: NGF 20 ng/ml (A,B), CNTF 10 ng/ml (C,O) and K t 35 mM (E.F). Magnificat ion 290 x . is unexpected and might point to differences in the intracellular mechanisms leading to neuronal survival in different neuronal populations. Thus, identifying the precise biochemical site and mode of action of K-252a could prove very useful for the eluci-dation of the signaltransduction of neurotrophic factors. Ithank Y. Matsuda a nd M. Sendtner for the generous gifts ofK-252a and CNTF. respectively, R. Heumann for helpful discussions, C. Krieger for excellent technical assistance and H . Thoenen for his supporl. Specia l thanks are due to Y.-A. Barde for many valuable suggestions and critical revision of the manuscript. The author was a recipient of a post-doctoral fellowship by the Deutsche Forschungsgemein-schaft (DFG). Aeheson. A.. vogl, W., Huuner. W.B. and Thocncn, H .. Methyltr.lnsferase inhibitors block NGF-regulatcd survi ... al and protein phosphorylation in sympathetic neurons. EMSO J .. 5 (1986) 2799- 2803. 2 Barbin. G .. Manthorpc. M. and Varon. 5 .. ruri/katiun of the ehk!:: eye ciliary neuronotrophie factor. J. Ncurochem .. 43( 1984) 1468- 1471\. 3 Barde. Y.·A .. What. if anylh ing. is a nturotrophie factor"!. T INS, I1 (1988) 343- 346. 4 Bar·Sagi. D. and Feramisco. 1.R .. Mieruinjcction uf the ras oneogene protein in to Pel2 cclls induces morphologieal differen tiation. Cell. 42 (1985) 841 - 848 . .5 Borasio, G.D .. 1ohn, 1 .. Wiuinghofer, A .. Barde. Y.-A .. Sendtner. M. and Heumann. R .. ras p2 1 pro-tein prOrnOtes survi ... al and fiber outgrowth of eul tured embryonie neurons. Neuron. 2 (1989) 1087- 1096. 6 Collins. F .• Axon initiation by eiliary neurons in eulture, Oe .... Biol., 6S (1978) 50-57. 7 DohcTly. P. and Walsh. F.S .. K-252a spccifically inhibits the survi ... al ;lnd morphologieal differentia-tion of NGF-dependent neurons in primary eultures of human dorsal rout ganglia. Neurosei. Lw .. 96 (1989) 1- 6. 8 Edgar. 0., Barde, Y.·A. and Thocnen. H .. Subpopulations of eultured sympathetie neurons differ in thcir rtquirements for $ur ... i ... al faetors. Nature (Lond.). 289 (198 1) 294-295. 9 Edgar, D .. TimpI. R. and Thoenen. H.. The hcparin-binding doma;n of laminin is responsible fur ftS effecls on neurile outgrowth and neuronal survi ... al, EMBO J .• 3 (1984)1463 1468. 10 Hagag, N .. Halegoua. S. and Viola, M .. Inhibition of growth faelor-induced differentiation or PC 12 cells by mieroinjection of antibody to ru.' p2 1. Nature (Lond.). 319 (1986) 680~2. 1I Hashimoto. 5., K-252a, a potent prote;n kinase inhibitor. blocks nerve growth faelor- induced neunte outgrowlh and ehanges in the phosphorylatiun of proteins in PCI2h edJs. J . Cell BioL. 107 (1988) 1531 - 1539. 12 Hofer. M.M. and Barde, Y.-A.. Brain-deri ... ed neurOlrophic factor prevents neuronal dea th in ... i ... o. Nature (Lond.). 331 (1988) 261 - 262. J3 Hughes. S.M .• Lillien. l.E .. Raff, M.C.. Robrer. H. and Sendtner. M .. Ciliary neurot rophie faetor in-duces type-2 astrocyte differentia tion in culture. Nature (Lond.), 335 (1988) 70-73. 14 Kasc. H .• lwahashi. K. and Matsuda, Y .• K-25la. a potent inhibitor or protein kinase C from mierobi-31 origin. J. Antibiot .. 39 (I 986) 1059 1065. 15 Koizumi. 5 .. Conlrer.ls. M.l.. Matsuda. Y .• Hilma. T., La7.aro ... ici , P. and Guroff. G., K-252a : a spe<;;-~e inhibitor of Ihe iletion of nerve growlh raelor on PC'12 cclls. J. Neurosei .. 8 (1988) 715 7~ I. 16 Lindsay, R.M .. Thocncn. H. and B;nde. Y.-A .. Plaeode and neural ercst·derived scnsorr neurons ilre responsi ... c ill earl)' developmental stages to brain-derived neurotrophie faelor, Oe .... Biol. . 112 (1985) 319 328. 17 Matsuda. Y. and Fukuda. J .. Inll ibition by K·252a, a new inhibitor 01' pro tein kinasc. ur nerve growlh faetor_induccd neurite outgrowth of ehiek embryo dors31 root gangli/)ß cclls. Neurosei . t etl.. X7 (198X) 11 (7. 18 Noda. M .. Ko, M., Ogura, A .. L,u. D .. Amanu. T. . Takano. T. and Ikawa. Y., Sarcoma virusesearry-ing ru.< oneogenes inducc differcntiation·assO/.;ated properlies in a neuronal eell line. Nature (Lond.). 318( 1985)7375. I<) Thocnen. H. and Barde, Y.-A .. Physiology ofner ... e growth factar, Physio1. Re ... .. 60 (1980) 121\4- 1335. 

Differential effects of the protein kinase inhibitor K-252a on the in vitro survival of chick embryonie neurons

Universität München: Elektronischen Publikationen

https://epub.ub.uni-muenchen.de/8826/1/differential_effects_of_the_protein_kinase_inhibitor_k-252a_on_the_vitro_survival_8826.pdf

Differential effects of the protein kinase inhibitor K-252a on the in vitro survival of chick embryonie neurons

Abstract

Similar works

Full text

Available Versions

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Universität München: Elektronischen Publikationen