Skip to main content
SpringerLink
Log in

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

  • Published:
Histochemistry Aims and scope Submit manuscript

Abstract

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC β, PIC γ, PIC δ. These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC β was detected in the nucleus whereas PIC δ was only cytoplasmic; PIC γ was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the γ isoform was concentrated near the nucleus, while the immunoreactivity of the β form remained constant and the δ form was increased. After 10 days of treatment with NGF, PIC β was hardly detectable and PIC γ immunostaining was considerably decreased. On the contrary, PIC δ progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC β and PIC γ were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC β and γ and the increase of PIC δ isozyme associated with the decrease of cell proliferation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Altin JG, Bradshaw RA (1990) Production of 1,2-diacylglycerol in PC12 cells by nerve growth factor and basic fibroblast growth factor. J Neurochem 54:1666–1676

    Google Scholar 

  • Arteaga CL, Johnson MD, Todderud G, Coffey RJ, Carpenter G, Page GL (1991) Elevated content of tyrosine kinase substrate phospholipase C-γ1 in primary human breast carcinomas. Proc Natl Acad Sci USA 88:10435–10439

    Google Scholar 

  • Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signalling. Nature 341:197–204

    Google Scholar 

  • Capitani S, Bertagnolo V, Mazzoni M, Santi P, Previati M, Antonucci A, Manzoli FA (1989) Lipid phosphorylation in isolated rat liver nuclei. FEBS Lett 254:194–198

    Google Scholar 

  • Capitani S, Mazzoni M, Bertagnolo V, Previati M, Daddona A, Ricci D, Manzoli FA (1990a) Nuclear inositol lipids in Friend erythroleukemia cells: changes related to differentiation induced by hexamethylenebisacetamide. Cell Biol Int Rep 14:783–795

    Google Scholar 

  • Capitani S, Helms B, Mazzoni M, Previati M, Bertagnolo V, Wirtz K, Manzoli FA (1990b) Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein. Biochim Biophys Acta 1044:193–200

    Google Scholar 

  • Capitani S, Billi AM, Bertagnolo V, Previati M, Mazzoni M, Neri LM, Manzoli FA (1991) Inositol lipid in Friend erythroleukemia cells: evidence for changes in nuclear metabolism after differentiation. Cell Biochem Funct 9:135–145

    Google Scholar 

  • Choi WC, Gerfen GR, Suh PG, Rhee SG (1989) Immunohistochemical localization of a brain isozyme of phospholipase C (PLC III) in astroglia rat brain. Brain Res 499:193–197

    Google Scholar 

  • Cocco L, Gilmour RS, Ognibene A, Lecther AJ, Manzoli FA, Irvine RF (1987) Synthesis of polyphosphoinositides in nuclei of Friend cells. Biochem J 248:765–770

    Google Scholar 

  • Contreras ML, Guroff G (1987) Calcium dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC12 cells. J Neurochem 48:1466–1472

    Google Scholar 

  • Cordon Cardo C, Tapley P, Jing S, Nanduri V, O'Rourke E, Lambelle F, Kovary K, Klein L, Jones KR, Reichardt LF, Barbacid M (1991) The trk tyrosine protein kinase mediates the mitogenic properties of NGF and neurotrophin-3. Cell 66:173–183

    Google Scholar 

  • Cremins J, Wagner JA, Halegona S (1986) Nerve growth factor action is mediated by cyclic AMP- and Ca2+ phospholipid dependent protein kinases. J Cell Biol 103:887–893

    Google Scholar 

  • Divecha N, Banfic H, Irvine RF (1991) The polyphosphoinositide cycle exists in the nuclei of Swiss 3T3 cells under the control of a receptor for IGF-I in the plasma membrane and stimulation of the cell cycle increases nuclear diacylglycerol and apparently induces translocation of protein kinase C. EMBO J 10:3207–3214

    Google Scholar 

  • Divecha N, Rhee SG, Lechter AJ, Irvine RF (1993) Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C β1 is specifically, but not predominantly, located in the nucleus. Biochem J 289:617–620

    Google Scholar 

  • Gerfen GR, Choi WC, Suh PG, Rhee SG (1988) Phospholipase C I and II brain isozymes: Immunohistochemical localization in neuronal systems in rat brain. Proc Natl Acad Sci USA 85:3208–3212

    Google Scholar 

  • Greene LA (1984) The importance of both early and delayed responses in the biological actions of nerve growth factor. Trends Neurosci 7:91–94

    Google Scholar 

  • Greene LA, Shooter EM (1980) The nerve growth factor: biochemistry, synthesis and mechanism of action. Annu Rev Neurosci 3:353–402

    Google Scholar 

  • Greene LA, Tischler AS (1976) Establishment of a nonadrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:2424–2428

    Google Scholar 

  • Gutowski S, Smrcka A, Nowak L, Wu DG, Simon M, Sternweis PC (1991) Antibodies to the α subfamily of guanine nucleotide-binding regulatory protein alpha subunits attenuate activation of phosphatidylinositol 4,5-bisphosphate hydrolysis by hormones. J Biol Chem 266:20519–20524

    Google Scholar 

  • Hama T, Huang KP, Guroff G (1986) Protein kinase C as a component of a nerve growth factor sensitive phosphorylation system in PC12 cells. Proc Natl Acad Sci USA 83:2353–2357

    Google Scholar 

  • Heasley LE, Johnson GL (1989) Regulation of protein kinase C by nerve growth factor, epidermal growth factor and phorbol esters in PC12 pheochromocytoma cells. J Biol Chem 264:8646–8652

    Google Scholar 

  • Hempstead BL, Martin Zanca D, Kaplan DR, Paroba LF, Chao MV (1991) High affinity NGF binding requires coexpression of the trk proto-oncogene and the low affinity NGF receptor. Nature 350:678–683

    Google Scholar 

  • Kato H, Fukami K, Shibasaki F, Homma Y, Takenawa T (1992) Enhancement of phospholipase C-δ1 activity in the aortas of spontaneously hypertensive rats. J Biol Chem 267:6483–6487

    Google Scholar 

  • Kim UH, Fink D, Kim HS, Park DJ, Contreras ML, Guroff G, Rhee SG (1991) Nerve growth factor stimulates phosphorylation of phospholipase C-γ in PC12 cells. J Biol Chem 266:1359–1362

    Google Scholar 

  • Klein R, Jing S, Nanduri V, O'Rowrkee E, Barbacid M (1991) The trk proto-oncogene encodes a receptor for NGF. Cell 65:189–197

    Google Scholar 

  • Kuriki H, Tamiya-Koizumi K, Asano M, Yoshida S, Kojima K, Nimura Y (1992) Existence of phosphoinositide-specific phospholipase C in rat liver nuclei and its change during liver regeneration. J Biochem 111:283–286

    Google Scholar 

  • Levi Montalcini R (1987) The nerve growth factor, thirty-five years later. EMBO J 6:1145–1154

    Google Scholar 

  • Maher PA (1988) Nerve growth factor induces protein tyrosine phosphorylation. Proc Natl Acad Sci USA 85:6788–6791

    Google Scholar 

  • Maraldi NM, Zini N, Squarzoni S, Del Coco R, Sabatelli P, Manzoli FA (1992) Intranuclear localization of phospholipids by ultrastructural cytochemistry. J Histochem Cytochem 40:1383–1392

    Google Scholar 

  • Martelli AM, Neri LM, Gilmour RS, Barker PJ, Huskisson NS, Manzoli FA, Cocco L (1991) Temporal changes in intracellular distribution of protein kinase C in Swiss 3T3 cells during mitogenic stimulation with insulin like growth factor I and bombesin: translocation to the nucleus follows rapid changes in nuclear polyphosphoinositides. Biochem Biophys Res Commun 177:480–487

    Google Scholar 

  • Martelli AM, Gilmour RS, Bertagnolo V, Neri LM, Manzoli L, Cocco L (1992) Nuclear localization and signalling activity of phosphoinositidase C β in Swiss 3T3 cells. Nature 358:242–244

    Google Scholar 

  • Martin-Zanca D, Oskam R, Mitra G, Copeland T, Barbacid M (1989) Molecular and biochemical characterization of the human trk proto-oncogene. Mol Cell Biol 9:24–33

    Google Scholar 

  • Mazzoni M, Bertagnolo V, Neri LM, Carini C, Milani D, Marchisio M, Manzoli FA, Capitani S (1992) Discrete subcellular localization of phosphoinositidase C β, γ and δ in PC12 rat pheochromocytoma cells. Biochem Biophys Res Commun 187:114–120

    Google Scholar 

  • McBride K, Rhee SG, Jaken S (1991) Immunocytochemical localization of phospholipase C γ in rat embryo fibroblasts. Proc Natl Acad Sci USA 88:7111–7115

    Google Scholar 

  • Meisenhelder J, Suh PG, Rhee SG, Hunter T (1989) Phospholipase C γ is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell 57:1109–1122

    Google Scholar 

  • Neri LM, Mazzotti G, Capitani S, Maraldi NM, Cinti C, Baldini N, Rana R, Martelli AM (1992) Nuclear matrix bound replicational sites detected in situ by 5-bromodeoxyuridine. Histochemistry 98:19–32

    Google Scholar 

  • Perrella FW (1990) Characterization of phosphatidylinositol phospholipase C activity in human melanoma. Biochem Biophys Res Commun 166:715–722

    Google Scholar 

  • Rana RS, Hokin LE (1990) Role of phosphoinositides in transmembrane signalling. Physiol Rev 70:115–164

    Google Scholar 

  • Rhee SG (1991) Inositol phospholipid specific phospholipase C: interaction of the γ1 isoform with tyrosine kinase. Trends Biochem Sci 16:297–301

    Google Scholar 

  • Rhee SG, Choi KD (1992) Regulation of inositol phospholipid specific phospholipase C isozymes. J Biol Chem 267:12393–12396

    Google Scholar 

  • Rhee SG, Suh PG, Ryu SH, Lee SY (1989) Studies of inositol phospholipid specific phospholipase C. Science 244:546–550

    Google Scholar 

  • Rydel RE, Greene LA (1987) Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci 7:3639–3653

    Google Scholar 

  • Shenker A, Goldsmith P, Unnson C, Spiegel A (1991) The G protein coupled to the tromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem 266:9309–9313

    Google Scholar 

  • Smith MR, Ryu SH, Suh P, Rhee SG, Kung H (1989) S-phase induction and transformation of quiescent NIH 3T3 cells by microinjection of phospholipase C. Proc Natl Acad Sci USA 86:3659–3663

    Google Scholar 

  • Smith MR, Liu Y, Kim H, Rhee SG, Kung H (1990) Inhibition of serum- and Ras-stimulated DNA synthesis by antibodies to phospholipase C. Science 247:1074–1077

    Google Scholar 

  • Smrcka AV, Heppler JR, Brown KO, Sternweis PC (1991) Regulation of phosphoinositide specific phospholipase C activity by purified Gq. Science 251:804–807

    Google Scholar 

  • Suh PG, Ryu SH, Choi WC, Lee KY, Rhee SG (1988) Monoclonal antibodies to three phospholipase C isozymes from bovine brain. J Biol Chem 263:14497–14504

    Google Scholar 

  • Taylor S, Chae H, Rhee SG, Exton J (1991) Activation of the β1 isozyme of phospholipase C by α subunits of the Gq class of G proteins. Nature 350:516–518

    Google Scholar 

  • Traynor AE, Shubert D, Allen WR (1982) Alterations of lipid metabolism in response to nerve growth factor. J Neurochem 39:1677–1683

    Google Scholar 

  • Vetter ML, Martin-Zanca D, Parada LF, Bishop JM, Kaplan DR (1991) Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-γ1 by a kinase activity associated with the product of the trk proto-oncogene. Proc Natl Acad Sci USA 88:5650–5654

    Google Scholar 

  • Voorhout WF, Genderen IL van, Yoshioka T, Fukami K, Geuze HJ, Meer G van (1992) Subcellular localization of glycolipids as revealed by immuno-electron microscopy. Trends Glycosci Glycotechnol 4:533–546

    Google Scholar 

  • Wu B, Lee Ch, Rhee SG, Simon MI (1992) Activation of phospholipase C by the α subunits of the Gq and G11 proteins in transfected cos-7 cells. J Biol Chem 267:1811–1817

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Istituto di Anatomia Umana Normale, Università di Ferrara, Via Fossato di Mortara 66, I-44100, Ferrara, Italy

    Luca M. Neri, Daniela Milani, Marco Marchisio, Lucia Bertolaso & Silvano Capitani

  2. Istituto di Citomorfologia Normale e Patologica del CNR, c/o Istituti Ortopedici Rizzoli, Via di Barbiano 1/10, I-40136, Bologna, Italy

    Luca M. Neri & Fiorenzo Marinelli

  3. Istituto Superiore di Sanita', Viale Regina Elena 299, I-00161, Roma, Italy

    Francesco A. Manzoli

Authors
  1. Luca M. Neri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniela Milani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marco Marchisio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lucia Bertolaso
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fiorenzo Marinelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Francesco A. Manzoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Silvano Capitani
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Neri, L.M., Milani, D., Marchisio, M. et al. Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation. Histochemistry 100, 121–129 (1993). https://doi.org/10.1007/BF00572898

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00572898

Keywords

Search

Navigation