Rat brain glial cells in primary culture and subculture contain the δ, ϵ and ζ subspecies of protein kinase C as well as the conventional subspecies

ELSEVIER

Neuroscience Letters 171 (1994) 117 120

N[UROSCI[NCE LETTERS

Rat brain glial cells in primary culture and subculture contain the d, e and subspecies of protein kinase C as well as the conventional subspecies Amanda L. Gott "**, Barbara S. Mallon a, Alex Paton b, Nigel Groome b, Martin G. Rumsby ~''* "Department ~?/'Biology, University o] York, }brk YOI 5DD, UK t~School ~/ Biological and Molecular Sciences. O.~/brd Brookes University, Oxlord 0~\:~ OBP. UK Received 10 September 1993; Revised version received 23 February 1994: Accepted 25 February 1994

Abstract

We raised polyclonal antibodies against the C-terminal peptides of protein kinase C (PkC) subspecies c~,15~,152, )', ~, ~, and ( and checked their specificity against brain extracts using Western immunoblot analysis. With equal amounts of protein applied to gels PkC subspecies fl~, ~, e and ~" were detected in primary cultures of mixed glial cells: bands for the c~ and 15~ subspecies were less prominent. PkC y was not detected in primary glial cultures. The e and ( subspecies of PkC were detected in subcultures of type 1 astrocytes with weaker bands for the ~z, 15~ and 152 subspecies. Blots of O-2A-lineage glia contained PkCs ~ and ( as prominent bands: the c¢./3~ and ~ subspecies were also present. All PkC subspecies including PkC y were detected in C6 glioma cells.

Key words: Protein kinase C subspecies: Primary glial cell culture: Type 1 astrocyte; O-2A lineage glia: Western blotting

The protein kinase C (PkC) family of calcium/phospholipid-dependent serine and threonine kinases is involved in the control of cellular functions such as transcription, secretion, growth and differentiation [9,19]. Ten PkC subspecies ~z. fl~, f12, )', ~, ~, O, ~1, ( and 2 have now been identified and their properties examined [9,19]. All PkC subspecies except 0 and r/ have been identified in brain tissue [19] and PkC y may be CNS specific [9]. PkC has been detected immunocytochemically in cells resembling oligodendrocytes [6,18] and PkC activity in primary cultures of astrocytes and mixed glial cells has been quantitated [4,13,15]. PkC ~z, but not PkC fl or y, has been detected in astrocytes but oligodendrocytes showed no immunoreactivity [20]. In optic nerve, PkC c¢ was detected in astrocytes [11]. PkC ~ was detected in all cells in primary cultures of rat glia, PkC fl~ was only found in type 2 astrocytes, microglia and oligodendrocytes while the ,B~ and ), subspecies were not detected [12]. m R N A for PkCs c~ and/3 was detected in *Corresponding author. Fax: (44) 904-432860.

**Present address: Chemistry and Cellular Sciences, SmithKlineBeecham, The Frythe, Welwyn, AL6 9AR, UK. 0304-3940/94/$7.00 ~? 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304- 3940(94)00187-F

oligodendrocytes developing in primary culture but PkC iF m R N A was expressed transiently [2]. Primary glial cell cultures and subcultures of O-2A lineage glia and type 1 astrocytes were prepared as described [13,21]. At 7 days, cultures consisted of a confluent monolayer of flat cells which showed phenotypic (GFAP +, A2B5 , GC-) and morphological characteristics of type 1 astrocytes. Small phase-bright processbearing cells appeared on the monolayer surface after some days and had a phenotype characteristic of O-2A progenitor glia (A2B5 +, G F A P , GC ) with some GC +, A2B5-, G F A P - cells, i.e. oligodendrocytes. Phase-bright cells shaken off the type 1 astrocyte monolayer and subcultured were a mixture of O-2A progenitor glia (A2B5 +, GC , GFAP-), type 2 astrocytes (A2B5 +, G F A P ÷, GC-) and oligodendrocytes (GC +, A2B5-, GFAW). Flat cells remaining after shake-off and subsequent passage were G F A P +, A2B5 , GC type 1 astrocytes. C-terminal peptides specific for rat or human brain PkC subspecies were synthesised with an N-terminal cysteine on an AMS 422 Multiple Peptide Synthesiser (Abimed, Langenfeld, Germany) using PyBop (benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate) as activator for Fmoc-amino acids [5].

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Amino acid sequences were as follows: ~, (C)PQFVHPILQSAV-COOH; fl~, (C)SEFLKPEVKS-COOH: f12, (C)NPEFVINV-COOH; 7, (C)PDARSPISPTPVPVMCOOH; 6, (C)VNPKYEQFLE-COOH; e,(C)NQEEFKGFSYFGEDLMP-COOH; ( (C)INPLLLSAEESVCOOH. Each peptide was examined by reverse phase HPLC and by plasma desorption mass spectrometry (University of Uppsala). In each case a strong major peak was observed on HPLC and a dominant molecular ion observed in the mass spectrum at the expected mass for each peptide. Peptides were coupled to keyhole limpet haemocyanin (Pierce) using malsac-HNSA [1]. New Zealand White rabbits were immunised initially with 500 /tg conjugated peptide in 2 ml Freund's complete adjuvant and were boosted twice at 4 week intervals with half the amount of conjugate. Rabbits were bled 2 weeks after the last boost. Cerebra from 1, 7, 16 and 42 day old rats were homogenised at a ratio of 100 /~g wet weight tissue/ml in 50 mM Tris/HCl pH 7.7, 2 mM EGTA and 5 mM dithiothreitol containing leupeptin (100 /tg/ml) and phenylmethylsulphonyl fluoride (2 raM). Primary cultures of glial cells at 4, 7 and 16 days and subcultures of type 1 astrocytes and of O-2A-lineage glia were scraped from flasks in 0,2 ml of homogenisation buffer, after rinsing the cell layer twice with cold Tris/saline. Brain and glial cell homogenates were diluted 2:1 with 10% SDS and then further diluted in SDS-PAGE solubilising buffer followed by heating at 100°C for 5 rain. Protein in homogenates was measured by the Pierce BCA method and equal amounts of protein were resolved by SDS-PAGE on 10% gels at a constant 200 V using a Bio-Rad minigel system. Proteins were transfered to nitrocellulose (S&S, 0.45/t) in a Bio-Rad Transblot system with Tris/glycine/methanol transfer buffer under standard conditions of 250 mA and 75 min. Blots were briefly stained with Ponceau S for 10 min to check transfer of proteins and destained in 1% acetic acid for immunoblotting. Blots were blocked at room temperature with 5% Marvel in 10 mM Tris-HCl, pH 7.5, 0.15 M NaCI (TBS) containing 0.2% Tween 20 for 1 h and then exposed to primary antibody at a dilution of l:1000 (~, fl~, f12, 6, and O and 1:150 (?9 in 1% Marvel in TBS for 18 h at room temperature. Blots were washed 3 times in TBS/ 0.1% Tween 20 and then incubated with anti-rabbit IgG/ peroxidase conjugate (Sigma), diluted l:10000 in TBS/ 1% Marvel for 1 h. Blots were rinsed 3 times in TBS/0.1% Tween 20 and peroxidase activity detected using DAB amplified by nickel solution (Vector Laboratories, Peterborough) or the ECL method (Amersham Int.). Amino acid sequences of the C-terminal peptides used to raise antisera were mostly for rat PkC which, for the 0~, fl~, f12 and e subspecies are identical with human sequences. Each PkC band on Western blots was competed out by preincubation of the antiserum with its appropriate peptide but not by peptides to other sequences con-

1994, / 1 7 /20

firming the specificity of the interaction. Antisera made to the same C-terminal peptide sequences have been found to recognise only their correct purified or recombinant PkC subspecies (P.J. Parker, personal communication and [16]). Using these antisera bands for PkC subspecies cz,/3~, f12, 6, e and ( were clearly identified at the correct molecular weight position in extracts of 42 day brain tissue (Fig. 1), PkC g having a higher molecular size [9,19]. PkC 3/was detected in mature brain extracts only (Fig. 1) and was not found in day I brain and was only weakly detected in day 7 brain samples (Fig. 4), in keeping with findings that this subspecies increases during brain development [10]. The antiserum to the PkC flz C-terminal peptide very often detected two bands, one band at the expected molecular size and the other at about 105 kDa: both were competed out with the specific peptide. The antiserum to the PkCc~ C-terminal peptide reacted with a component of molecular size on blots at about 97 kDa (Figs. 1, 2, 3 and 4) which was not competed out with
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Fig. I. Detection of protein kinase C subspecies in cerebral total proteins from 42 day rat brain by Western blot analysis with DAB staining using polyclonal antibodies raised to the PkC C-terminal peptides described. For each pair of blots the left hand lane is with antibody alone and the right hand lane with antibody blocked with the appropriate peptide. Experimental conditions are described in the text: markers are 116, 97.4, 66, 45 and 29 kDa. Arrows indicate positions of faint f12 and ( subspecies visible on blots.

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PkC ~ was present in all glial cell preparations in agreement with Masliah et al. [12] and both/3~ and f12 subspecies were present in mixed glial cultures and in type 1 astrocytes. Of the two splice variants, the/3~ subspecies seemed to be the mai~n form, however (B.S. Mallon, unpublished observations) and in O-2A lineage glia, the fl~ subspecies is the only variant readily detected. Detection of both/3~ and/32 subspecies in the same primary glial cultures does not mean that both splice variants occur in the sa~ne cell because primary glial cultures and O-2A progenitor glia subcultures are heterogeneous in cell composition. Type 1 astrocytes in subculture have a common GFAP +, GC-, A2B5- phenotype but may still be heterogeneous as shown by a differing response to certain ligands [17]. PkC ?' seems to be absent from glia, in agreement with other findings [11,12] and its detection in whole brain extracts (Fig. 1) suggests that this subspecies is either specific for non-glial cells of the CNS or is expressed at some later developmental stage as found t\~r PkC~' m R N A in oligodendrocyte-enriched glial cultures [2]. In all our analyses we detected PkC ?" in C6 glioma cells and the antibody response was competed out with the ?" subspecies specific C-terminal peptide but not with other peptides. PkC is central to signal transduction

Fig. 2. Western blot analyses with DAB staining of protein kinase C subspecies in A, 4 day and B, 11 day primary glial cell cultures. The right hand lane in each pair is with anti C-terminal peptide antibody blocked with the specific C-terminal peptide. Molecular weight markers for A (right) are at 116, 97, 65 45, 35 and 29 kDa and for B (left) are 116, 97, 66, 45 and 29 kDa. Arrows indicate PkC subspecies detected.

fainter bands for the ~, 6 and fl~ subspecies (Fig. 3A). With O-2A lineage glia (type 2 astrocytes, oligodendrocytes and O-2A progenitor glia) prominent bands for PkCs 6"and ~"were detected with weaker bands for PkCs and fl~ also being observed (Fig. 3B). Using the sensitive ECL method (Fig. 4) PkC e was confirmed in subcultures of type 1 astrocytes while PkC 6" was a prominent subspecies in O-2A lineage glia but was detected more faintly in type 1 astrocytes. Both type 1 astrocytes and O-2A lineage glia contained PkC ~. Prominent bands indicating the presence of the 6, g and ~" subspecies of PkC were detected in C6 cells with the ~ and/3~ forms present as well. With our anti-PkC 7 C-terminal peptide antiserum we routinely detect PkC ~' in C6 glioma cells (Fig. 4) but not in primary glia. PkC ~' was detected only faintly in the 7 day post-natal brain samples used in ECL blots (Fig. 4, ~'). Our results reveal the presence of PkCs 6., g and ( in primary glial cell cultures, at the times selected for analysis, with the PkC ~ and/3 subspecies also present. The affinity of each antiserum for its target protein is not known and therfore band intensity on Western blots cannot be taken to indicate subspecies levels present.

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Fig. 3. Western blot analyses with DAB staining of PkC subspecies in subcultures of type 1 astrocytes (A) and of shake-off 0 2A lineage glia (B). Right hand lanes of each pair are with antibody blocked by the specific C-terminal peptide. Markers t\~r A Iright) are at 116, 97.4, 65 45, 35 and 29 kDa and for B (left) are 116, 97.4, 66, 45 and 29 kDa. Arrows indicate PkC bands detected.

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Fig. 4. Western blot analyses with ECL development for PkC ~ (~), PkC 7/(),), PkC ~ (o") and PkC e (e) in C6 glioma cells (C6), type 1 astrocytes (As), O-2A progenitor glia (02) and 7 day brain samples (Br). Marker bars are at 116, 97.4, 66 and 45 kDa in c~and 7/and 116, 97.4 and 66 kDa in d~and e.

pathways regulating glial cell growth [7,22], differentiation [3], proliferation [4,8,14,15] and process formation [23]. It is now necesssary to elucidate the role of individual PkC subspecies in regulating glial cell growth and

development. This work was funded by grants from the Multiple Sclerosis Society of Britain and Northern Ireland, The Wellcome Trust and the University of York Innovation Fund. We thank Rebecca Turner for excellent cell culture work. [1] Aldwin, L. and Nitecki, D.E., A water-soluble, monitorable peptide and protein cross-linking agent, Anal. Biochem., 164 (1987) 494~501. [2] Asotra, K. and Macklin, W.B., Protein kinase C activity modulates myelin gene expression in enriched oligodendrocytes, J. Neurosci. Res., 34 (1993) 571-588. [3] Avossa, D. and Pfeiffer, S.E., Transient reversion of 04" GalC- oligodendrocyte progenitor development in response to the phorbol ester TPA, J. Neurosci. Res., 34 (1993) 113-128. [4] Bhat, N.R., Role of protein kinase C in glial cell proliferation, J. Neurosci. Res., 22 (1989) 20-27. [5] Gausepohl, H., Kraft, M., Boulin, C. and Frank, R.W., Automated multiple peptide synthesis with BOP activation. In J.E. Rivier and G.R. Marshall (Eds.), Peptides: Chemistry Structure and Biology, ESCOM, Leiden, 1990, pp, 1003-1004. [6] Girard, RR., Mazzei, G.J., Wood J.G. and Kuo, J.F., Polyclonal antibodies to phospholipid/Ca2+-dependent protein kinase and immunocytochemical localisation of the enzyme in rat brain, Proc. Natl. Acad. Sci. USA, 82 (1985) 3030-3034.

[7] Hart, I.K., Richardson, WD.. Bolsover. S.R. and Raft. M . ( , PDGF and intracellular signalling in the timing of oligodendrocyte differentiation, J. Cell Biol., 109 (1989) 3411 341"7,. [8] Honneger, R, Protein kinase C-activating tmnour promoters enhance the differentiation of astrocytes in aggregating foetal brain cell cultures, J. Neurochem., 46 (1986) 1561 1566. [9] Hug, H. and Sarre, T.F., Protein kinase C isoenzymes: divergence in signal transduction?, Biochem. J., 291 (I993) 329 343. [10] Kikkawa, U., Ogita, K., Shearman, M.S., Ase, K., Sekiguchi, K_ Naor, Z., Ido, M., Nishizuka, Y., Saito, N., Tanaka, C., Ono, Y., Fujii, T. and Igarashi, K., The heterogeneity and differential expression of protein kinase C in nervous tissues, Phil. Trans. R. Soc. Lond. B, 320 (1988) 313.324. [11] Komoly, S., Liu, Y., Webster, tt. deF. and Chan, K.-F. J.. Distribution of protein kinase C isozymes in ral optic nerves, J. Neurosci. Res., 29 (1991) 379 389. [121 Masliah, E., Yoshida, K., Shimohama, S., Gage, F. and Saitoh, T,, Differential expression of protein kinase C isozymes in rat glial cell cultures, Brain Res., 549 (1991) 106-111. [13] Murphy, J.A., Turner, R. M. and Rumsby, M.G., The protein kinase C, calcium-independent and calcium-dependent kinase activities of glial cells in primary culture and subculture, Neurochem. Int., 23 (1993) 87-94. [14] Murphy, S., McCabe, N., Morrow, C. and Pearce, B.R., Phorbot ester stimulates proliferation ofastrocytes in primary culture, Dev. Brain Res., 31 (1987) 133-135. [15] Neary, J.T., Norenberg, L.O.B. and Norenberg, M.D., Protein kinase C in primary astrocyte cultures: cytoplasmic localisation and translocation by a phorbol ester, J. Neurochem., 50 (1988) 1179 1184. [16] Olivier, A.R. and Parker, RJ., Identification of multiple PKC isoforms in Swisss 3T3 cells: differential down-regulation by phorbol ester, J. Cell. Physiol., 152 (1992) 240-244. [17] Shao, Y., Enkvist, K. and McCarthy, K., Astroglial adrenergic receptors, in S. Murphy (Ed.), Astrocytes, Pharmacology and Function, Academic Press, New York, 1993, pp. 25 45. [18] Shimohama, S., Saito, T. and Gage, F.H., Differential expression of protein kinase C isozymes in rat cerebellum, J. Chem. Neuroanat., 3 (1990) 367 375. [19] Stabel, S. and Parker, RJ., Protein kinase C, Pharmac. Ther., 51 (1991) 71-95. [20] Todo, T., Shitara, N., Nakamura, H., Takakura, K., Tomonaga, M. and Ikeda, K., Astrocytic localisation of the immunoreactivity for protein kinase C isozyme (type III) in human brain, Brain Res., 517 (1990) 351-353. [21] Walker, A.G., Chapman, J.A., Bruce, C.B. and Rumsby, M.G., Immunocytochemical characterisation of cell cultures grown from dissociated 1- 2 day post-natal rat cerebral tissue, J. Neuroimmunol., 7 (1985) 1-20. [22] Williams, L.T., Signal transduction by the platelet-derived growth factor receptor, Science, 243 (1989) 1564-1570. [23] Yong, V.W., Cheung, J.C.B., Uhm, J.H. and Kim, S.U., Agedependent decrease of process formation by cultured oligodendrocytes is augmented by protein kinase C stimulation, J. Neurosci. Res., 29 (1991) 87 99.