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Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4)

  • Marcelo Coutinho de Miranda
    Footnotes
    Affiliations
    Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627 Belo Horizonte–MG, 31270-901, Brazil

    Section of Digestive Diseases, Internal Medicine, Yale University, New Haven, Connecticut 06520-8056
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  • Michele Angela Rodrigues
    Footnotes
    Affiliations
    Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627 Belo Horizonte–MG, 31270-901, Brazil

    Section of Digestive Diseases, Internal Medicine, Yale University, New Haven, Connecticut 06520-8056
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  • Ana Carolina de Angelis Campos
    Footnotes
    Affiliations
    Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627 Belo Horizonte–MG, 31270-901, Brazil

    Section of Digestive Diseases, Internal Medicine, Yale University, New Haven, Connecticut 06520-8056
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  • Jerusa Araújo Quintão Arantes Faria
    Footnotes
    Affiliations
    Department of Physiology Sciences, Universidade Federal do Amazonas, Manaus-AM, 69080-900, Brazil
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  • Marianna Kunrath-Lima
    Affiliations
    Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627 Belo Horizonte–MG, 31270-901, Brazil
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  • Gregory A. Mignery
    Affiliations
    Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
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  • Deborah Schechtman
    Affiliations
    Department of Biochemistry, University of São Paulo, Av. Professor Lineu Prestes, 748, São Paulo–SP 05508-900, Brazil
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  • Alfredo Miranda Goes
    Affiliations
    Department of Pathology, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627 Belo Horizonte–MG, 31270-901, Brazil
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  • Michael H. Nathanson
    Affiliations
    Section of Digestive Diseases, Internal Medicine, Yale University, New Haven, Connecticut 06520-8056
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  • Dawidson A. Gomes
    Correspondence
    To whom correspondence should be addressed: Instituto de Ciências Biológicas, Bloco Q4, Sala 238, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627 Belo Horizonte–MG, Brazil 31270-901
    Affiliations
    Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos, 6627 Belo Horizonte–MG, 31270-901, Brazil

    Section of Digestive Diseases, Internal Medicine, Yale University, New Haven, Connecticut 06520-8056
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  • Author Footnotes
    4 Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
    1 These authors contributed equally to this work.
    3 The abbreviations used are:ERendoplasmic reticulumAVParginine vasopressinCHC2clathrin heavy chain 2CFPcyan fluorescent proteinDAGdiacylglycerolEGFepidermal growth factorEGFREGF receptorFRETFörster resonance energy transferInsP3inositol 1,4,5-trisphosphateInsP3RInsP3 receptormRFPmonomeric red fluorescent proteinNRnucleoplasmic reticulumPdBuphorbol 12–13-dibutyratePI(4,5)P2phosphatidylinositol 4,5-bisphosphatePKCprotein kinase CPLCphospholipase CRTKreceptor tyrosine kinaseYFPyellow fluorescent proteinHGFhepatocyte growth factorANOVAanalysis of variance.
Open AccessPublished:September 19, 2019DOI:https://doi.org/10.1074/jbc.RA118.006961
      Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

      Introduction

      The spatial-temporal distribution of calcium (Ca2+) signals contributes to the versatility of this second messenger (
      • Berridge M.J.
      The inositol trisphosphate/calcium signaling pathway in health and disease.
      ). For example, increases in Ca2+ within the cell nucleus selectively promote cellular events such as gene transcription (
      • Pusl T.
      • Wu J.J.
      • Zimmerman T.L.
      • Zhang L.
      • Ehrlich B.E.
      • Berchtold M.W.
      • Hoek J.B.
      • Karpen S.J.
      • Nathanson M.H.
      • Bennett A.M.
      Epidermal growth factor-mediated activation of the ETS domain transcription factor Elk-1 requires nuclear calcium.
      ), cell proliferation, and tumor growth (
      • Rodrigues M.A.
      • Gomes D.A.
      • Leite M.F.
      • Grant W.
      • Zhang L.
      • Lam W.
      • Cheng Y.-C.
      • Bennett A.M.
      • Nathanson M.H.
      Nucleoplasmic calcium is required for cell proliferation.
      ). Moreover, nuclear Ca2+ signals can be regulated independently of cytosolic Ca2+ signals (
      • Hardingham G.E.
      • Chawla S.
      • Johnson C.M.
      • Bading H.
      Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression.
      ). This is possible because the nucleus contains the machinery necessary for Ca2+ mobilization (
      • Echevarría W.
      • Leite M.F.
      • Guerra M.T.
      • Zipfel W.R.
      • Nathanson M.H.
      Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.
      • Gomes D.A.
      • Leite M.F.
      • Bennett A.M.
      • Nathanson M.H.
      Calcium signaling in the nucleus.
      ,
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ). Specifically, the nuclear envelope is contiguous with the endoplasmic reticulum (ER)
      The abbreviations used are: ER
      endoplasmic reticulum
      AVP
      arginine vasopressin
      CHC2
      clathrin heavy chain 2
      CFP
      cyan fluorescent protein
      DAG
      diacylglycerol
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      FRET
      Förster resonance energy transfer
      InsP3
      inositol 1,4,5-trisphosphate
      InsP3R
      InsP3 receptor
      mRFP
      monomeric red fluorescent protein
      NR
      nucleoplasmic reticulum
      PdBu
      phorbol 12–13-dibutyrate
      PI(4,5)P2
      phosphatidylinositol 4,5-bisphosphate
      PKC
      protein kinase C
      PLC
      phospholipase C
      RTK
      receptor tyrosine kinase
      YFP
      yellow fluorescent protein
      HGF
      hepatocyte growth factor
      ANOVA
      analysis of variance.
      (
      • Subramanian K.
      • Meyer T.
      Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores.
      ) and has invaginations that can reach deep within the nucleoplasm (
      • Malhas A.
      • Goulbourne C.
      • Vaux D.J.
      The nucleoplasmic reticulum: form and function.
      ). These invaginations express InsP3Rs, store and release Ca2+ in an InsP3-sensitive fashion (
      • Echevarría W.
      • Leite M.F.
      • Guerra M.T.
      • Zipfel W.R.
      • Nathanson M.H.
      Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.
      ,
      • Stehno-Bittel L.
      • Lückhoff A.
      • Clapham D.E.
      Calcium release from the nucleus by InsP3 receptor channels.
      ), and have been referred to as the nucleoplasmic reticulum (NR) (
      • Echevarría W.
      • Leite M.F.
      • Guerra M.T.
      • Zipfel W.R.
      • Nathanson M.H.
      Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.
      ,
      • Malhas A.
      • Goulbourne C.
      • Vaux D.J.
      The nucleoplasmic reticulum: form and function.
      ). The distribution of InsP3R isoforms and also Ca2+ signaling in the nucleus become altered in various disease states, such as fatty liver disease (
      • Khamphaya T.
      • Chukijrungroat N.
      • Saengsirisuwan V.
      • Mitchell-Richards K.A.
      • Robert M.E.
      • Mennone A.
      • Ananthanarayanan M.
      • Nathanson M.H.
      • Weerachayaphorn J.
      Nonalcoholic fatty liver disease impairs expression of the type II inositol 1,4,5-trisphosphate receptor.
      ) and cholangiocarcinoma (
      • Ueasilamongkol P.
      • Khamphaya T.
      • Guerra M.T.
      • Rodrigues M.A.
      • Gomes D.A.
      • Kong Y.
      • Wei W.
      • Jain D.
      • Trampert D.C.
      • Ananthanarayanan M.
      • Banales J.M.
      • Roberts L.R.
      • Farshidfar F.
      • Nathanson M.H.
      • Weerachayaphorn J.
      Type 3 inositol 1,4,5-trisphosphate receptor is increased and enhances malignant properties in cholangiocarcinoma.
      ). Intranuclear InsP3 is formed from PI(4,5)P2 hydrolysis, which is induced by growth factors such as hepatocyte growth factor (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ) and insulin (
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ). Some RTKs undergo nuclear translocation upon activation, which appears necessary for initiation of nuclear Ca2+ signals (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ), so this could be one mechanism to regulate Ca2+ release locally.
      Nuclear localization of EGFR is of clinical relevance, as it is correlated with cancer prognosis (
      • Brand T.M.
      • Iida M.
      • Li C.
      • Wheeler D.L.
      The nuclear epidermal growth factor receptor signaling network and its role in cancer.
      ) and relates to resistance to various cancer therapies (
      • Li C.
      • Iida M.
      • Dunn E.F.
      • Ghia A.J.
      • Wheeler D.L.
      Nuclear EGFR contributes to acquired resistance to cetuximab.
      ,
      • Huang W.C.
      • Chen Y.J.
      • Li L.Y.
      • Wei Y.L.
      • Hsu S.C.
      • Tsai S.L.
      • Chiu P.C.
      • Huang W.P.
      • Wang Y.N.
      • Chen C.H.
      • Chang W.C.
      • Chang W.C.
      • Chen A.J.
      • Tsai C.H.
      • Hung M.C.
      Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells.
      ). Nuclear EGFR function has been the subject of extensive investigation, as has been nuclear targets of EGFR. EGFR can shuttle from the cell surface to the nucleus (
      • De Angelis Campos A.C.
      • Rodrigues M.A.
      • de Andrade C.
      • de Goes A.M.
      • Nathanson M.H.
      • Gomes D.A.
      Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway.
      ,
      • Faraco C.C.F.
      • Faria J.A.Q.A.
      • Kunrath-Lima M.
      • Miranda M.C.
      • de Melo M.I.A.
      • Ferreira A.D.F.
      • Rodrigues M.A.
      • Gomes D.A.
      Translocation of epidermal growth factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
      ) where it acts as a transcriptional regulator (
      • Lin S.Y.
      • Makino K.
      • Xia W.
      • Matin A.
      • Wen Y.
      • Kwong K.Y.
      • Bourguignon L.
      • Hung M.C.
      Nuclear localization of EGF receptor and its potential new role as a transcription factor.
      ,
      • Huo L.
      • Wang Y.N.
      • Xia W.
      • Hsu S.C.
      • Lai C.C.
      • Li L.Y.
      • Chang W.C.
      • Wang Y.
      • Hsu M.C.
      • Yu Y.L.
      • Huang T.H.
      • Ding Q.
      • Chen C.H.
      • Tsai C.H.
      • Hung M.C.
      RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.
      ), transmits signals (
      • Wang S.C.
      • Nakajima Y.
      • Yu Y.L.
      • Xia W.
      • Chen C.T.
      • Yang C.C.
      • McIntush E.W.
      • Li L.Y.
      • Hawke D.H.
      • Kobayashi R.
      • Hung M.C.
      Tyrosine phosphorylation controls PCNA function through protein stability.
      ,
      • Liccardi G.
      • Hartley J.A.
      • Hochhauser D.
      EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment.
      ), and is involved in processes such as cell proliferation, tumor progression, and DNA repair and replication (
      • Wang Y.N.
      • Hung M.C.
      Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family.
      ). However, it is unknown whether EGFR is linked to nuclear calcium release.
      PLC could be involved in primary nuclear Ca2+ signaling because activation of InsP3 receptors generally is involved in the release of Ca2+ in the nucleus (
      • Bootman M.D.
      • Fearnley C.
      • Smyrnias I.
      • MacDonald F.
      • Roderick H.L.
      An update on nuclear calcium signalling.
      ). In mammals, the PLC family is composed of 13 isozymes, divided into six classes as follows: β, γ, δ, ɛ, ζ, and η, according to their structures (
      • Nakamura Y.
      • Fukami K.
      Regulation and physiological functions of mammalian phospholipase C.
      ). All these isozymes catalyze the reaction of PI(4,5)P2 cleavage, generating InsP3 and diacylglycerol (DAG), but each one possesses unique physiological functions (
      • Nakamura Y.
      • Fukami K.
      Regulation and physiological functions of mammalian phospholipase C.
      ). Some PLCs are described within the nucleus in specific cell types such as PLCβ1, PLCγ1, PLCδ1, PLCδ4, and PLCζ (
      • Faenza I.
      • Fiume R.
      • Piazzi M.
      • Colantoni A.
      • Cocco L.
      Nuclear inositide specific phospholipase C signalling–interactions and activity.
      ,
      • Leung D.W.
      • Tompkins C.
      • Brewer J.
      • Ball A.
      • Coon M.
      • Morris V.
      • Waggoner D.
      • Singer J.W.
      Phospholipase C δ-4 overexpression upregulates ErB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells.
      ). The activity of these nuclear PLCs is related to the regulation of several cellular processes, such as proliferation and differentiation. These particular isoforms also have been linked to specific diseases, including myelodysplastic syndromes (PLCβ1), neurological diseases (PLCγ1), and infertility (PLCζ and PLCδ1–4) (
      • Ratti S.
      • Mongiorgi S.
      • Ramazzotti G.
      • Follo M.Y.
      • Mariani G.A.
      • Suh P.G.
      • McCubrey J.A.
      • Cocco L.
      • Manzoli L.
      Nuclear inositide signaling via phospholipase C.
      ).
      PLCδ4 has been identified as a nuclear protein in different cell types and may be involved in proliferative processes (
      • Leung D.W.
      • Tompkins C.
      • Brewer J.
      • Ball A.
      • Coon M.
      • Morris V.
      • Waggoner D.
      • Singer J.W.
      Phospholipase C δ-4 overexpression upregulates ErB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells.
      ,
      • Lee S.B.
      • Rhee S.G.
      Molecular cloning, splice variants, expression, and purification of phospholipase C-δ4.
      ,
      • Nagano K.
      • Fukami K.
      • Minagawa T.
      • Watanabe Y.
      • Ozaki C.
      • Takenawa T.
      A novel phospholipase C δ4 (PLCδ4) splice variant as a negative regulator of PLC.
      • Kunrath-Lima M.
      • de Miranda M.C.
      • Ferreira A.D.F.
      • Faraco C.C.F.
      • de Melo M.I.A.
      • Goes A.M.
      • Rodrigues M.A.
      • Faria J.A.Q.A.
      • Gomes D.A.
      Phospholipase Cδ4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.
      ). It was first purified from regenerating rat liver protein extracts (
      • Asano M.
      • Tamiya-Koizumi K.
      • Homma Y.
      • Takenawa T.
      • Nimura Y.
      • Kojima K.
      • Yoshida S.
      Purification and characterization of nuclear phospholipase C specific for phosphoinositides.
      ), and its gene was cloned (
      • Liu N.
      • Fukami K.
      • Yu H.
      • Takenawa T.
      A new phospholipase Cδ4 is induced at S-phase of the cell cycle and appears in the nucleus.
      ) from a regenerating rat liver cDNA library, indicating a possible role of PLCδ4 in cell proliferation. Despite its nuclear localization, it is unknown how human PLCδ4 is activated within the nucleus. This work investigates whether EGFR activates nuclear Ca2+ signaling via PLCδ4.

      Results

      EGFR translocates to the nucleus and induces Ca2+ signals

      Stimulation with EGF induces its RTK EGFR to translocate to the nucleus (
      • Faraco C.C.F.
      • Faria J.A.Q.A.
      • Kunrath-Lima M.
      • Miranda M.C.
      • de Melo M.I.A.
      • Ferreira A.D.F.
      • Rodrigues M.A.
      • Gomes D.A.
      Translocation of epidermal growth factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
      ), similar to what has been shown for other RTKs, including the hepatocyte growth factor (HGF) receptor, c-Met, and the insulin receptor (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ). EGFR translocated to the nucleus in both primary rat hepatocytes and SKHep-1 cells, a human liver cancer cell line (Fig. 1A). EGFR protein levels in the nuclear fraction were higher than in nonstimulated control cells as soon as 2.5 min after stimulation with EGF (Fig. 1A). In SKHep-1 cells, EGFR decreased in the non-nuclear fraction as it increased in the nuclear fraction. The peak of EGFR nuclear translocation in this cell line was at 10 min (Fig. 1A). Super-resolution immunofluorescence showed that EGFR accumulated in the nucleus within 10 min of exposure to EGF (Fig. 1B). EGF stimulation induced bigger EGFR clusters throughout the cells, including at the nucleus, as demonstrated previously (
      • Li C.
      • Iida M.
      • Dunn E.F.
      • Ghia A.J.
      • Wheeler D.L.
      Nuclear EGFR contributes to acquired resistance to cetuximab.
      ). These data demonstrate that EGF induces EGFR to translocate to the nucleus.
      Figure thumbnail gr1
      Figure 1EGF induces EGFR nuclear translocation and clathrin-mediated endocytosis-dependent Ca2+ signals. A, Western blot analysis of EGFR in non-nuclear and nuclear fractions isolated from resting (control) or EGF-stimulated hepatocyte and SKHep-1 cells. α-Tubulin and lamin B1 were used as purity controls for non-nuclear and nuclear fractions, respectively. Indicated kDa values represent the position of the referenced bands from pre-stained protein standard. Densitometric measurements of cellular fractions show that nuclear EGFR is maximal within 10 min of stimulation in SKHep-1 cells and within 2.5–5 min (p < 0.01) in hepatocytes when compared with the control (0 min). Values are scaled relative to the initial amount in the non-nuclear fraction and relative to the amount at 2.5 min in the nuclear fraction (mean ± S.E.; n = 3) (nuclear SKHep-1 fractions, one-way ANOVA, F(4,10) = 3.215; p = 0.0611; nuclear hepatocyte fractions, one-way ANOVA, F(4,10) = 8.530, p = 0.0029). B, gated STED super-resolution images of control SKHep-1 cells (right image) and 10 min after EGF stimulation (middle image). Serial optical sections were collected for three-dimensional reconstruction; y-z sections are shown at the right of each image. EGFR is represented in green; lamin B1 is in red, and the nuclear compartment marked in blue. Note that EGFR redistributes to the region of the nuclear envelope as well as within the nuclear interior (arrows). Bar = 10 μm. Scatter plot (right panel) showing the quantification of EGFR that co-localizes with the nucleus before and after EGF stimulation. n = 7–12 cells for each group. The image collection settings for fluorescence quantification was adjusted according to the cells stimulated with EGF to avoid nuclear-saturated pixels of EGFR clusters. ***, p < 0.001 (Student’s t test). C, Western blotting (top) analysis of clathrin heavy chain 2 (CHC2) expression in nontreated, Lipofectamine only, control siRNAs-treated, or CHC2 siRNAs-treated SKHep-1 cells. α-Tubulin was used as a loading control. Bar graph shows the summary of the Western blottings (n = 4); ***, p < 0.001. (One-way ANOVA, F(5,18) = 34.41; p < 0.0001.) D, tracings represent Fluo-4/AM fluorescence intensity changes, normalized by the baseline, by the time of EGF stimulation. Lower panel, bar graph compiling peaks of Fluo-4/AM fluorescence intensity in EGF stimulated at 15 min, control siRNA-treated (n = 15), Lipofectamine only (n = 14), control siRNA 1 (n = 10), control siRNA 2 (n = 10), CHC2 siRNA 1 (n = 13), or CHC2 siRNA 2-treated (n = 13) SKHep-1 cells. Fluorescence changes over time from whole cells were normalized and represented as fluorescence intensity (F) by baseline fluorescence (F0) multiplied by 100. ***, p < 0.001. (One-way ANOVA, F(5,69) = 18.78; p < 0.0001.) Experiments were performed on at least 3 different days.
      Translocation of EGFR to the nucleus depends on clathrin-mediated endocytosis (
      • De Angelis Campos A.C.
      • Rodrigues M.A.
      • de Andrade C.
      • de Goes A.M.
      • Nathanson M.H.
      • Gomes D.A.
      Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway.
      ), so we used clathrin heavy chain 2 (CHC2) siRNAs to disrupt this endocytic pathway and thereby inhibit internalization of EGFR. Fig. 1C shows that siRNA treatment reduced CHC2 expression by 94 ± 3% using the CHC2 siRNA 1 and by 87 ± 8% using the CHC2 siRNA 2, relative to control (p < 0.001). Stimulation of control cells with EGF led to a gradual Ca2+ increase with some superimposed oscillations, similar to the Ca2+ signal pattern induced by other growth factors (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ), but CHC2 knockdown diminished the peak of EGF-induced Ca2+ signals by 85 ± 2% (p < 0.001) using the CHCH2 siRNA 1 and by 100 ± 2% (p < 0.001) using the CHCH2 siRNA 2, compared with control (Fig. 1D). This indicates that most of the Ca2+ signals induced by EGF depend on EGFR internalization.

      EGF triggers intranuclear PI(4,5)P2 hydrolysis and InsP3 formation

      EGF induces intracellular Ca2+ transients through InsP3 (
      • Moccia F.
      • Berra-Romani R.
      • Tritto S.
      • Signorelli S.
      • Taglietti V.
      • Tanzi F.
      Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells.
      ), so we investigated whether EGF-induced InsP3 formation in the nucleus was responsible for increasing intranuclear Ca2+. SKHep-1 cells were transfected with cytosolic or nuclear InsP3-buffer constructs whose targeting was confirmed by subcellular localization of the monomeric red fluorescent protein (mRFP) (Fig. 2A). Fluo-4/AM was used to monitor Ca2+ release in response to EGF. Cytosolic InsP3-buffer decreased the Ca2+ response by 50.5 ± 6.1% in the nucleus and by 53.8 ± 4.5% in the cytoplasm (Fig. 2B), but EGF-induced Ca2+ signals were decreased by 86 ± 2.7% in the nucleus and by 96 ± 3.9% in the cytoplasm in cells expressing the nuclear InsP3-buffer (Fig. 2B). This suggests that EGF triggers nuclear Ca2+ signals by inducing the formation of InsP3 within the nucleus, similar to what has been observed in liver cells stimulated with HGF (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ) or insulin (
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ). To investigate whether EGF induces nuclear PI(4,5)P2 hydrolysis to produce InsP3, PI(4,5)P2 was measured in nuclear fractions of hepatocytes. Nuclei from EGF-stimulated cells contained 64 ± 1.5% less PI(4,5)P2 than nuclei from control cells (Fig. 2D). These findings provide evidence that EGF triggers nuclear PI(4,5)P2 hydrolysis and local InsP3 production to generate Ca2+ signals.
      Figure thumbnail gr2
      Figure 2EGF triggers Ca2+ release by nuclear InsP3 and nuclear PI(4,5)P2 hydrolysis. A, confocal images of SKHep-1 cells loaded with Fluo-4/AM expressing cytosolic InsP3-buffer, nuclear InsP3-buffer, or mRFP (control). Images show cells before EGF stimulus (baseline) and the peak (15 min) of EGF-induced Fluo-4/AM fluorescence intensity changes. Expression of constructs was checked by detection of mRFP. Bar = 10 μm. B, bar graph showing Fluo-4/AM fluorescence intensity peak in cytosolic and nuclear regions of control, cytosolic, or nuclear InsP3-buffer expressing cells upon EGF stimulus (8–11 cells in each group: *, p < 0.05; **, p < 0.01; and ***, p < 0.001) (cytosol: one-way ANOVA, F(2,24) = 17.17, p < 0.0001; nuclear, one-way ANOVA, F(2,26) = 28.03; p < 0.0001.) C, average traces of SKHep-1 expressing cytosolic or nuclear InsP3-buffer or control cells stimulated with EGF. Cytosolic or nuclear regions for fluorescent intensity measurements were selected using the assistance of the digital image of contrast (D.I.C) images, see squares at the nucleus and non-nuclear regions. Nuclear but not cytosolic InsP3-buffer blocked the peak of EGF response in both compartments. D, bar graph represents the amount of PI(4,5)P2 of nucleus isolated from control or EGF-stimulated hepatocytes (n = 6). 5 min of EGF stimulation reduces nuclear PI(4,5)P2 by 64 ± 1.5% (p < 0.05) (Student’s t test).

      EGF stimulates intra-nuclear PKC activity

      PI(4,5)P2 hydrolysis generates not only InsP3 but also DAG, which can activate PKC (
      • Bootman M.D.
      • Fearnley C.
      • Smyrnias I.
      • MacDonald F.
      • Roderick H.L.
      An update on nuclear calcium signalling.
      ). Increases in nuclear DAG can either participate in translocation of PKCs, from the cytosol to the nucleus, or can directly activate PKCs that reside in the nucleus (
      • Martelli A.M.
      • Evangelisti C.
      • Nyakern M.
      • Manzoli F.A.
      Nuclear protein kinase C.
      ). To determine whether EGF triggers nuclear PKC activity, we used a Förster resonance energy transfer (FRET) reporter based on PKC activity and tagged to a nuclear localization signal (NucCKAR) (
      • Gallegos L.L.
      • Kunkel M.T.
      • Newton A.C.
      Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
      ). The nuclear localization of this construct was confirmed by intra-nuclear detection of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fluorescence by confocal microscopy (Fig. 3A). Phosphorylation of the reporter by PKC results in decreased rather than increased FRET (
      • Gallegos L.L.
      • Kunkel M.T.
      • Newton A.C.
      Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
      ), so the CFP/YFP ratio was monitored to reflect PKC activity. NucCKAR-expressing SKHep-1 cells were stimulated with EGF, phorbol 12–13-dibutyrate (PdBu) as a positive control for PKC activation, or arginine vasopressin (AVP) to reflect cytosolic activation of PKC, and then changes in FRET were followed for 30 min (Fig. 3B). PdBu increased CFP/YFP emission of NucCKAR (Fig. 3, B and C), and similar results were obtained with EGF treatment (Fig. 3, B and C), indicating that either stimulus increases nuclear PKC activity. Unlike EGF, stimulation with AVP, which activates a prototypical G-protein–coupled receptor in the plasma membrane, did not increase the CFP/YFP emission ratio of NucCKAR (Fig. 3, B and C). This result suggests that subplasmalemmal formation of DAG in response to AVP does not activate nuclear PKC. Treatment with the broad-range PKC inhibitor Gö6983 (Fig. 3D) decreased the EGF-induced CFP/YFP emission ratio, providing additional evidence that EGF triggers PKC activity within the nucleus.
      Figure thumbnail gr3
      Figure 3EGF induces nuclear PKC activity. A, confocal images of a representative SKHep-1 cell expressing NucCKAR shows CFP and YFP emission as well as a transmitted image of the cell, confirming nuclear localization of the construct. Bar = 10 μm. B, representative tracings of NucCKAR CFP/YFP emission normalized by baseline show changes in emission ratio during 30 min of perfusion with 200 nm phorbol 12–13-dibutyrate (PdBu) (n = 6), 100 ng/ml EGF (n = 10), or 500 nm AVP (n = 3). C, bar graph compiling the average results shown in B. NucCKAR FRET ratio change in EGF-stimulated cells in the presence (n = 8) or absence (n = 10) of the PKC inhibitor Gö6983 (250 nm). The PKC inhibitor significantly attenuates EGF-induced NucCKAR FRET changes: *, p < 0.05 (one-way ANOVA, F(3,23) = 5.645; p = 0.0002).

      PLCγ1 is localized to the cytosol and PLCδ4 is in the nucleus

      PLC mediates PI(4,5)P2 hydrolysis (
      • Ratti S.
      • Mongiorgi S.
      • Ramazzotti G.
      • Follo M.Y.
      • Mariani G.A.
      • Suh P.G.
      • McCubrey J.A.
      • Cocco L.
      • Manzoli L.
      Nuclear inositide signaling via phospholipase C.
      ) and formation of InsP3 and DAG (
      • Lee S.B.
      • Rhee S.G.
      Molecular cloning, splice variants, expression, and purification of phospholipase C-δ4.
      ,
      • Nagano K.
      • Fukami K.
      • Minagawa T.
      • Watanabe Y.
      • Ozaki C.
      • Takenawa T.
      A novel phospholipase C δ4 (PLCδ4) splice variant as a negative regulator of PLC.
      ), so the relative role played by several candidate PLC isoforms during EGF stimulation was investigated. PLCγ1 is the primary isoform typically thought to bind to and be activated by RTKs (
      • Berridge M.J.
      The inositol trisphosphate/calcium signaling pathway in health and disease.
      ). PLCδ4 has been recognized as a predominantly nuclear PLC isoform, and its expression is increased in liver regeneration (
      • Liu N.
      • Fukami K.
      • Yu H.
      • Takenawa T.
      A new phospholipase Cδ4 is induced at S-phase of the cell cycle and appears in the nucleus.
      ), so we compared the localization of these two isoforms in SKHep-1 cells. Confocal immunofluorescence showed that PLCγ1 is mostly localized to the cytosol, and this pattern does not change upon EGF stimulation (Fig. 4A). In contrast, PLCδ4 was detected exclusively in the nucleus of SKHep-1 cells, demonstrated by both confocal microscopy (Fig. 4B) and Western blotting (data not shown). The subcellular localization of these PLC isoforms was also assessed in primary rat hepatocytes. Similar to what was observed in SKHep-1 cells, PLCγ1 was found in non-nuclear fractions of both control and EGF-stimulated cells, whereas PLCδ4 was only detected in the nuclear fractions (Fig. 4C). The amount of PLCγ1 or PLCδ4 in each cell fraction was not altered by stimulation with EGF (Fig. 4C), indicating that there is no translocation of these PLCs between nuclear and non-nuclear compartments in response to EGF. Further examination showed that EGF stimulation for up to 10 min does not induce PLCγ1 to translocate from the cytoplasm to the nucleus (Fig. 4D).
      Figure thumbnail gr4
      Figure 4PLCγ1 is a cytosolic and PLCδ4 is a nuclear protein. A, representative confocal images of SKHep-1 cells before and after EGF stimulation showing PLCγ1 in red, EGFR in green, and TO-PRO nuclear dye in blue. Merged images reveal that PLCγ1 is predominantly expressed outside of the nucleus in both conditions (n = 15 cells). B, representative confocal images of control and EGF-stimulated SKHep-1 cells showing PLCδ4 in red, which is exclusively intra-nuclear (n = 15 cells). C, Western blotting showing PLCγ1 and PLCδ4 expressions in non-nuclear and nuclear fractions from control or EGF-stimulated hepatocytes. α-Tubulin and lamin B1 were used as purity controls for nuclear and non-nuclear fractions. Indicated kDa values represent the position of the reference bands from pre-stained protein standard. Subcellular localizations of PLCγ1 and PLCδ4 in hepatocytes are not altered by EGF stimulation for 10 min (n = 3). D, tracings of normalized amounts of PLCγ1 in non-nuclear and nuclear fractions of SKHep-1 cells upon EGF stimulation with different time points, quantified from Western blotting experiments (n = 3).

      PLCδ4 participates in EGF-induced nuclear PI(4,5)P2 hydrolysis, InsP3 signaling, Ca2+ signals, and PKC activity

      An siRNA approach was employed to determine the relative roles of PLCδ4 and PLCγ1 in EGF-induced nuclear PI(4,5)P2 hydrolysis and Ca2+ signaling. Specific siRNAs reduced PLCγ1 expression by 90 ± 14 and 91 ± 27% using siRNAs 1 and 2, respectively. PLCδ4 expressions were reduced by 75 ± 7 and 86 ± 5% using siRNAs 1 and 2, respectively (p < 0.01) (Fig. 5A). Nuclear fractions were isolated from these cells after EGF stimulation, and PI(4,5)P2 was quantified. EGF significantly increased nuclear PI(4,5)P2 hydrolysis in control siRNAs and PLCγ1 siRNA-treated cells, but not in PLCδ4 siRNA-treated cells (Fig. 5B). Furthermore, cells treated with PLCδ4 siRNAs significantly reduced nuclear InsP3 production, nuclear PKC activity, and Ca2+ signals, but this was not seen in PLCγ1 siRNA-treated cells (Fig. 5, C–E). These results suggest that PLCδ4 but not PLCγ1 participates in nuclear PI(4,5)P2 hydrolysis, InsP3 production, PKC activation, and Ca2+ signaling triggered by EGF stimulation.
      Figure thumbnail gr5
      Figure 5Intra-nuclear PLCδ4 mediates EGF-induced nuclear PI(4,5)P2 depletion and Ca2+ signals. A, Western blotting showing PLCγ1 and PLCδ4 expression in SKHep-1 cells under control, Lipofectamine, control siRNAs, siRNAs for PLCδ4, or PLCγ1 siRNAs-treated conditions. α-Tubulin was used as a loading control. The molecular mass of each protein analyzed is on the right side. Bar graphs show the summary of the Western blottings. Specific siRNAs reduced PLCγ1 expression by 90 ± 14 and 91 ± 27% using siRNAs 1 and 2, respectively (one-way ANOVA, F(7,16) = 26.60; p < 0.001). PLCδ4 expressions were reduced by 75 ± 7 and 86 ± 5% using siRNAs 1 and 2, respectively (**, p < 0.01) (one-way ANOVA, F(7,32) = 5.808; p = 0.0002). ns, not significant. PLCγ1 siRNAs do not alter each PLCδ4 expression level or PLCδ4 siRNAs do not alter each PLCγ1 expression levels. B, graph shows PI(4,5)P2 quantification in nuclei isolated from SKHep-1 cells before (−EGF group) and after 10 min of stimulation with EGF (+EGF groups). EGF-induced PI(4,5)P2 hydrolysis was blocked by PLCδ4 siRNAs, but not by PLCγ1 siRNAs, compared with cells not treated with EGF (−EGF). Observe that the −EGF group has no difference with PLCδ4 siRNA-treated cells, or PLCγ1 has no difference with EGF-treated cells. n = 3. **, p < 0.01 (one-way ANOVA F(8,18) = 88.26; p < 0.0001). C, left: representative average tracings of nuclear InsP3 sensor (FIRE-1nuc) shows changes in YFP/CFP emission normalized by baseline in EGF-stimulated groups over 20 min. Smoothing filter was used to better discern the tracings. Right: bar graph compiling the average results shows peak intensity in control (n = 11), control siRNA 1 (n = 13), control siRNA 2 (n = 13), PLCδ4 siRNA 1 (n = 11), PLCδ4 siRNA 2 (n = 12), PLCγ1 siRNA 1 (n = 11), or PLCγ1 siRNA 2-treated (n = 11) SKHep-1 cells. InsP3 production is significantly reduced by PLCδ4 siRNAs (100 ± 0.5% for both siRNAs, *, p < 0.05), but not by PLCγ1 siRNAs, compared with control groups (one-way ANOVA, F(7,93) = 6.717; p < 0.0001). D, left: representative average tracings of NucCKAR CFP/YFP emission normalized by baseline shows changes in emission ratio during 25 min of perfusion with EGF. Smoothing filter was used to better discern the tracings. Right: bar graph compiling the average results shows peak intensity in control (n = 12), control siRNA 1 (n = 11), control siRNA 2 (n = 9), PLCδ4 siRNA 1 (n = 7), PLCδ4 siRNA 2-treated (n = 7), PLCγ1 siRNA 1 (n = 6), or PLCγ1 siRNA 2-treated (n = 8) SKHep-1 cells. PKC activation is significantly reduced by PLCδ4 siRNAs (100.7 ± 0.4% for siRNA 1 and 100 ± 0.7% for siRNA 2, *, p < 0.05), but not by PLCγ1 siRNAs, compared with control groups (Kruskal-Wallis, H = 37.85, df = 7, p < 0,0001; Dunn’s multiple comparisons test). E, left: average tracings represent Fluo-4/AM fluorescence intensity changes in response to EGF stimulation. Fluorescence changes over time from whole cells were normalized and are represented as fluorescence intensity (F) divided by baseline fluorescence (F0), multiplied by 100%. Right: bar graph compiling the average results shows peak intensity in control (n = 20), Lipofectamine (n = 21), control siRNA 1 (n = 13), control siRNA 2 (n = 10), PLCδ4 siRNA 1 (n = 13), PLCδ4 siRNA 2-treated (n = 10), PLCγ1 siRNA 1 (n = 5), or PLCγ1 siRNA 2-treated (n = 9) SKHep-1 cells. Ca2+ is significantly reduced by PLCδ4 siRNAs (95.9 ± 2.5% for siRNA 1 and 96.9 ± 2.6% for siRNA 2, *, p < 0.05) but not by PLCγ1 siRNAs, compared with control groups (one-way ANOVA, F(7,92) = 15.66; p < 0.0001).

      PLCδ4 participates in cell cycle progression

      Nuclear Ca2+ is important for cell proliferation (
      • Rodrigues M.A.
      • Gomes D.A.
      • Leite M.F.
      • Grant W.
      • Zhang L.
      • Lam W.
      • Cheng Y.-C.
      • Bennett A.M.
      • Nathanson M.H.
      Nucleoplasmic calcium is required for cell proliferation.
      ), an important downstream effect of EGF. Therefore, we investigated whether PLCδ4 is involved in EGFR-mediated cell proliferation. Cell proliferation as measured by either cell counting (Fig. S1) or BrdU incorporation (Fig. 6A) was decreased by over 67% in cells in which PLCδ4 expression was reduced. SKHep-1 proliferation was restored when PLCδ4 expression was recovered (Fig. S1). Cell death does not mediate the decreased proliferation because annexin V–FITC and propidium iodide assays did not reveal cell death between control and siRNA-treated groups (Fig. 6B). Next, the role of PLCδ4 in cell cycle progression was investigated. To understand the basis for this, the effects of PLCδ4 on the expression of various cyclins were studied (Fig. S1). Expression of cyclin B1, a G2/M-phase checkpoint protein, and cyclin A, an S-phase checkpoint protein, was decreased in PLCδ4 knockdown cells compared with cells treated with control siRNA (Fig. 6C). A nuclear InsP3-buffer also decreased the expression of cyclins A and B. These findings suggest that nuclear PLCδ4 affects cell cycle progression, in part by affecting cyclin expression.
      Figure thumbnail gr6
      Figure 6Knockdown of PLCδ4 diminishes cell growth and decreases cyclins A and B1 without affecting cell death. A, effects of PLCδ4 depletion on cell proliferation. BrdU measurements were performed 48 h after transfection. *, p < 0.05 (EGF−, one-way ANOVA, F(5,35) = 8.042; p < 0.0001; EGF+, one-way ANOVA, F(5.35) = 9.570; p < 0.0001). B, quantification of cells labeled with annexin V-FITC and propidium iodide. The bar graph shows the percentages of cells labeled with either annexin V-FITC or propidium iodide or double-labeled with annexin V-FITC plus propidium iodide compared with all cells from each field. Each treatment was made in triplicate, and for each one, three different images were counted using ImageJ software; then, the means were used for statistical analysis. All groups were compared with control groups (control, Lipofectamine, and control siRNAs). Doxorubicin (Dox) 10 μg/ml was used as positive control, and it differs from all experimental groups. ***, p < 0.001. Annexin V staining did not reveal differences in the level of cell death between control and siRNA-treated groups (one-way ANOVA, F(6,71) = 20.64; p < 0.0001). C, Western blotting showing cyclins A and B1 expression in SKHep-1 cells under controls or siRNA-treated conditions. α-Tubulin was used as a loading control. Representative image of three independent experiments. Middle and right graphs, quantification of the Western blottings of cyclins that were normalized with the expression of α-tubulin (n = 3). *, p < 0.05, and ***, p < 0.001 (cyclin A, one-way ANOVA, F(6,14) = 17.92; p < 0.0001; cyclin B1, one-way ANOVA, F(5,12) = 69.17; p < 0.0001). D, Western blotting showing the effects of nuclear InsP3 buffer on the expression of the cyclins A and B1. Right graph, quantification of the Western blottings of cyclins that were normalized with the expression of α-tubulin (n = 3). **, p < 0.01 (cyclin A and B1 used a Student’s t test).

      Discussion

      EGFR has several direct effects on signaling in the nucleus, including interaction with transcription factors (
      • Shi Y.
      • Tao Y.
      • Jiang Y.
      • Xu Y.
      • Yan B.
      • Chen X.
      • Xiao L.
      • Cao Y.
      Nuclear epidermal growth factor receptor interacts with transcriptional intermediary factor 2 to activate cyclin D1 gene expression triggered by the oncoprotein latent membrane protein 1.
      ) and phosphorylation (
      • Wang S.C.
      • Nakajima Y.
      • Yu Y.L.
      • Xia W.
      • Chen C.T.
      • Yang C.C.
      • McIntush E.W.
      • Li L.Y.
      • Hawke D.H.
      • Kobayashi R.
      • Hung M.C.
      Tyrosine phosphorylation controls PCNA function through protein stability.
      ). This work extends this repertoire of intranuclear actions by showing that EGFR translocates to the nucleus of hepatic cells to initiate InsP3-mediated Ca2+ signals. EGFR likely translocates from the plasma membrane to the nucleus via the ER (
      • Huo L.
      • Wang Y.N.
      • Xia W.
      • Hsu S.C.
      • Lai C.C.
      • Li L.Y.
      • Chang W.C.
      • Wang Y.
      • Hsu M.C.
      • Yu Y.L.
      • Huang T.H.
      • Ding Q.
      • Chen C.H.
      • Tsai C.H.
      • Hung M.C.
      RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.
      ,
      • Wang Y.N.
      • Hung M.C.
      Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family.
      ), and a similar trafficking route has been described for both viruses and toxins as well (
      • Sandvig K.
      • van Deurs B.
      Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin.
      ,
      • Marsh M.
      • Helenius A.
      Virus entry: open sesame.
      • Carpenter G.
      • Liao H.J.
      Trafficking of receptor tyrosine kinases to the nucleus.
      ). Trafficking of other RTKs from the plasma membrane to the nucleus has also been described. For example, c-Met that is biotinylated at the plasma membrane can be recovered from the nucleus after cells are stimulated with HGF (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ). Similarly, fluorescently-labeled EGF can be tracked from the cell membrane to the cell nucleus over a 10-min period (
      • Faraco C.C.F.
      • Faria J.A.Q.A.
      • Kunrath-Lima M.
      • Miranda M.C.
      • de Melo M.I.A.
      • Ferreira A.D.F.
      • Rodrigues M.A.
      • Gomes D.A.
      Translocation of epidermal growth factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
      ). Furthermore, super-resolution imaging can be used to quantify the amount of EGF/EGFR clusters that accumulate in the nucleus (
      • Faraco C.C.F.
      • Faria J.A.Q.A.
      • Kunrath-Lima M.
      • Miranda M.C.
      • de Melo M.I.A.
      • Ferreira A.D.F.
      • Rodrigues M.A.
      • Gomes D.A.
      Translocation of epidermal growth factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
      ). The peak in nuclear translocation of EGFR coincides with the peak in Ca2+ signals (Fig. 1, A–C). This work builds on the previous observation that translocation of EGFR to the nucleus depends on dynamin and clathrin-mediated endocytosis (
      • De Angelis Campos A.C.
      • Rodrigues M.A.
      • de Andrade C.
      • de Goes A.M.
      • Nathanson M.H.
      • Gomes D.A.
      Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway.
      ) by showing that knockdown of clathrin reduces EGF-induced Ca2+ signals. These findings are consistent with the idea that EGFR must translocate to the nucleus in order to initiate nuclear Ca2+ signals, similar to what has been shown for c-Met (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ).
      Ca2+ signaling in the nucleus rather than cytosol is important for proliferation and tumor growth (
      • Rodrigues M.A.
      • Gomes D.A.
      • Leite M.F.
      • Grant W.
      • Zhang L.
      • Lam W.
      • Cheng Y.-C.
      • Bennett A.M.
      • Nathanson M.H.
      Nucleoplasmic calcium is required for cell proliferation.
      ). Nuclear Ca2+ can also regulate gene expression (
      • Pusl T.
      • Wu J.J.
      • Zimmerman T.L.
      • Zhang L.
      • Ehrlich B.E.
      • Berchtold M.W.
      • Hoek J.B.
      • Karpen S.J.
      • Nathanson M.H.
      • Bennett A.M.
      Epidermal growth factor-mediated activation of the ETS domain transcription factor Elk-1 requires nuclear calcium.
      ,
      • Hardingham G.E.
      • Chawla S.
      • Johnson C.M.
      • Bading H.
      Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression.
      ). Thus, increases in nuclear Ca2+ could be one mechanism for EGF to promote those cellular processes. The nucleus contains the machinery necessary for InsP3 receptor-mediated Ca2+ release (
      • Echevarría W.
      • Leite M.F.
      • Guerra M.T.
      • Zipfel W.R.
      • Nathanson M.H.
      Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.
      ,
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ,
      • Bootman M.D.
      • Fearnley C.
      • Smyrnias I.
      • MacDonald F.
      • Roderick H.L.
      An update on nuclear calcium signalling.
      ). Although PLC-mediated PI(4,5)P2 hydrolysis and formation of InsP3 and DAG within the cytoplasm have been described extensively (
      • Berridge M.J.
      The inositol trisphosphate/calcium signaling pathway in health and disease.
      ), there are fewer studies showing that PI(4,5)P2 hydrolysis also occurs within the nucleus. This work provides evidence that EGF triggers PI(4,5)P2 hydrolysis and InsP3 formation within the nucleus (Figure 2, Figure 5), similar to what has been observed in response to stimulation with HGF (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ) or insulin (
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ).
      PLC within the nucleus is involved in signal-transduction pathways that are separate from those activated by isoforms localized in other compartments (
      • Follo M.Y.
      • Finelli C.
      • Mongiorgi S.
      • Clissa C.
      • Bosi C.
      • Testoni N.
      • Chiarini F.
      • Ramazzotti G.
      • Baccarani M.
      • Martelli A.M.
      • Manzoli L.
      • Martinelli G.
      • Cocco L.
      Reduction of phosphoinositide-phospholipase C β1 methylation predicts the responsiveness to azacitidine in high-risk MDS.
      ). The most common paradigm links PLCβ isoforms with G-protein–coupled receptors, whereas RTKs typically are associated with PLCγ isoforms (
      • Nishibe S.
      • Wahl M.I.
      • Hernández-Sotomayor S.M.
      • Tonks N.K.
      • Rhee S.G.
      • Carpenter G.
      Increase of the catalytic activity of phospholipase C-γ1 by tyrosine phosphorylation.
      ). For example, the activated nuclear metabotropic glutamate 5 (mGlu5) receptor couples to Gq/11 and a nuclear isoform, PLCβ1, to generate the InsP3-mediated release of Ca2+ from Ca2+-release channels in the nucleus of primary striatal neurons (
      • Kumar V.
      • Jong Y.J.
      • O'Malley K.L.
      Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release.
      ). This work provides evidence that EGF induces hydrolysis of nuclear PI(4,5)P2 by the intra-nuclear PLCδ4, rather than by PLCγ1 (Fig. 5). It remains unclear whether PLCδ4 is activated directly or indirectly, as a direct binding of PLCδ4 with nuclear EGFR could not be demonstrated by co-immunoprecipitation (data not shown).
      There are a number of previous studies about the function of nuclear PLCs. Some studies suggest that PLC positively regulates the cell cycle. PLCβ1 can regulate insulin-like growth factor-1–stimulated DNA synthesis, and its overexpression increases proliferation in the absence of mitogenic stimuli (
      • Manzoli L.
      • Billi A.M.
      • Rubbini S.
      • Bavelloni A.
      • Faenza I.
      • Gilmour R.S.
      • Rhee S.G.
      • Cocco L.
      Essential role for nuclear phospholipase Cβ1 in insulin-like growth factor I-induced mitogenesis.
      ,
      • Faenza I.
      • Ramazzotti G.
      • Bavelloni A.
      • Fiume R.
      • Gaboardi G.C.
      • Follo M.Y.
      • Gilmour R.S.
      • Martelli A.M.
      • Ravid K.
      • Cocco L.
      Inositide-dependent phospholipase C signaling mimics insulin in skeletal muscle differentiation by affecting specific regions of the cyclin D3 promoter.
      ). Furthermore, PLCβ1 in the nucleus regulates transcriptional levels of genes such as cyclin D3 and c-jun activation in skeletal muscle cells (
      • Faenza I.
      • Ramazzotti G.
      • Bavelloni A.
      • Fiume R.
      • Gaboardi G.C.
      • Follo M.Y.
      • Gilmour R.S.
      • Martelli A.M.
      • Ravid K.
      • Cocco L.
      Inositide-dependent phospholipase C signaling mimics insulin in skeletal muscle differentiation by affecting specific regions of the cyclin D3 promoter.
      ,
      • Ramazzotti G.
      • Faenza I.
      • Gaboardi G.C.
      • Piazzi M.
      • Bavelloni A.
      • Fiume R.
      • Manzoli L.
      • Martelli A.M.
      • Cocco L.
      Catalytic activity of nuclear PLC-β1 is required for its signalling function during C2C12 differentiation.
      ). This study demonstrates that PLCδ4 also plays a role in cellular proliferation and specifically in cell-cycle control. Knockdown of PLCδ4 leads to decreased cell proliferation without affecting apoptosis in SKHep-1 cells (Fig. 6). The cell cycle profile is accompanied by reduced expression of cyclins A and B1 (Fig. 6). Consistent with this, decreased cyclin B1 could interfere with the transition through the G2/M phase of the cell cycle. Furthermore, it was demonstrated that PLCδ4 knockdown impaired human mesenchymal stem/stromal cells proliferation, without inducing cell death (
      • Kunrath-Lima M.
      • de Miranda M.C.
      • Ferreira A.D.F.
      • Faraco C.C.F.
      • de Melo M.I.A.
      • Goes A.M.
      • Rodrigues M.A.
      • Faria J.A.Q.A.
      • Gomes D.A.
      Phospholipase Cδ4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.
      ).
      The direct target of PLC signaling is typically PKC, which is activated by the PI(4,5)P2 hydrolysis products DAG and InsP3 (
      • Martelli A.M.
      • Evangelisti C.
      • Nyakern M.
      • Manzoli F.A.
      Nuclear protein kinase C.
      ). Because EGF activates PKC within the nucleus (Fig. 3), and several nuclear proteins are PKC substrates, this may also be relevant for processes such as gene expression and changes in chromatin structure (
      • Huang W.
      • Mishra V.
      • Batra S.
      • Dillon I.
      • Mehta K.D.
      Phorbol ester promotes histone H3-Ser10 phosphorylation at the LDL receptor promoter in a protein kinase C-dependent manner.
      ). Activation of PKC may affect cell cycle progression as well (
      • Parekh D.B.
      • Ziegler W.
      • Parker P.J.
      Multiple pathways control protein kinase C phosphorylation.
      ,
      • Fima E.
      • Shtutman M.
      • Libros P.
      • Missel A.
      • Shahaf G.
      • Kahana G.
      • Livneh E.
      PKCη enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.
      ). PKCα is necessary for PLCβ1-mediated regulation of cyclin D3 and cell proliferation in human erythroleukemia cells (
      • Poli A.
      • Faenza I.
      • Chiarini F.
      • Matteucci A.
      • McCubrey J.A.
      • Cocco L.
      K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway.
      ), and PKCα regulates cyclin B1 leading to effects on G2/M transition. PKC inhibitors can decrease cyclin B1 expression consequent to PKC down-regulation (
      • Poli A.
      • Ramazzotti G.
      • Matteucci A.
      • Manzoli L.
      • Lonetti A.
      • Suh P.G.
      • McCubrey J.A.
      • Cocco L.
      A novel DAG-dependent mechanism links PKCα and cyclin B1 regulating cell cycle progression.
      ). The increase in nuclear DAG level observed during the G2 phase is sufficient to activate nuclear PKCα/PKCβ. PKC phosphorylates lamin B1 at sites involved in mitotic disassembly of the nuclear lamina, one of the most critical events in the G2/M transition (
      • Fiume R.
      • Ramazzotti G.
      • Teti G.
      • Chiarini F.
      • Faenza I.
      • Mazzotti G.
      • Billi A.M.
      • Cocco L.
      Involvement of nuclear PLCβ1 in lamin B1 phosphorylation and G2/M cell cycle progression.
      ).
      In light of these considerations, the current findings identify novel signaling events that may link EGFR nuclear translocation to stimulation of cell growth (Fig. 7). Activation of this signaling pathway may, in turn, be important for hepatocyte proliferation, especially because liver regeneration is absolutely dependent on the presence of either EGF or HGF (
      • Michalopoulos G.K.
      Hepatostat: liver regeneration and normal liver tissue maintenance.
      ). This is also consistent with the observation that PLCδ4 expression is increased in liver regeneration as well as in liver cancer cell lines (
      • Liu N.
      • Fukami K.
      • Yu H.
      • Takenawa T.
      A new phospholipase Cδ4 is induced at S-phase of the cell cycle and appears in the nucleus.
      ,
      • Akutagawa A.
      • Fukami K.
      • Banno Y.
      • Takenawa T.
      • Kannagi R.
      • Yokoyama Y.
      • Oda K.
      • Nagino M.
      • Nimura Y.
      • Yoshida S.
      • Tamiya-Koizumi K.
      Disruption of phospholipase Cδ4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C.
      ). Furthermore, it is consistent with data from PLCδ4 knockout mice that show increases in bromodeoxyuridine (BrdU) are delayed after partial hepatectomy, although liver regeneration is not delayed (
      • Akutagawa A.
      • Fukami K.
      • Banno Y.
      • Takenawa T.
      • Kannagi R.
      • Yokoyama Y.
      • Oda K.
      • Nagino M.
      • Nimura Y.
      • Yoshida S.
      • Tamiya-Koizumi K.
      Disruption of phospholipase Cδ4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C.
      ). Therefore, strategies to selectively inhibit PLCδ4 could be important for selective inhibition of liver cancer, which is the third leading cause of cancer death worldwide.
      Figure thumbnail gr7
      Figure 7Proposed model: EGF triggers nuclear PI(4,5)P2 hydrolysis, local InsP3 production, and Ca2+ release into the nucleoplasm through intra-nuclear PLCδ4, which mediates cell proliferation. Scheme of the proposed EGF-induced nuclear Ca2+ signaling pathway. Upon EGF binding EGFR is activated and initiates signal transducer cascades. EGFR is shuttled to the nucleus to induce downstream targets. PLCδ4 is a nuclear enzyme induced by EGF stimulus and leads to PI(4,5)P2 hydrolysis that generates InsP3 and DAG. InsP3 binds to InsP3R present on the inner nuclear envelope triggering Ca2+ release in the nucleoplasm. DAG can activate nuclear-resident PKCs. Ca2+ and PKCs can activate downstream targets which ultimately result in cell proliferation.

      Experimental procedures

      Cell culture and EGF stimulus

      The SKHep-1 is a human liver cancer cell line (ATCC, Manassas, VA). It was cultured at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) containing 10% fetal bovine serum, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Inc.).
      Hepatocytes were isolated from healthy male Sprague-Dawley rats (190–200 g; Charles River Laboratories), as described previously (
      • Boyer J.L.
      • Phillips J.M.
      • Graf J.
      Preparation and specific applications of isolated hepatocyte couplets.
      ). Primary cells were cultured at 37 °C in 5% CO2 in Williams’ medium E (Life Technologies, Inc.) containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin and plated on collagen-coated coverslips (50 μg/ml) (BD Biosciences). Hepatocytes were used within 24 h of isolation. All animal studies were approved by the Yale University Institutional Animal Care and Use Committee under Protocol no. 2018-07602. Serum-starved (14–16 h) cells were treated with 100 ng/ml human recombinant EGF (Life Technologies, Inc.) or recombinant rat EGF (R&D Systems) at 37 °C in 5% CO2.

      Subcellular fractionation and Western blotting

      Cells were washed twice with ice-cold 10 mm PBS, pH 7.4 (PBS) (Sigma), harvested by scraping, and lysed in a buffer with 20 mm HEPES, pH 7.0, 10 mm KCl, 2 mm MgCl2, 0.5% Nonidet P-40. After incubation on ice for 10 min, the cells were homogenized by vortex. The homogenate was centrifuged at 1500 × g for 5 min to sediment the nuclei. The supernatant was then centrifuged at a maximum speed of 16,100 × g for 20 min, and the resulting supernatant formed the non-nuclear fraction. The nuclear pellet was washed three times with lysis buffer to remove any contamination from cytoplasmic membranes. The isolated nuclei were resuspended in NETN buffer (150 mm NaCl, 1 mm EDTA, 20 mm Tris-HCl, pH 8.0, 0.5% Nonidet P-40), and the mixture was sonicated briefly to aid nuclear lysis. Nuclear lysates were collected after centrifugation at 16,100 × g for 20 min at 4 °C. Protease and phosphatase inhibitors (Sigma) were added to all buffers. Proteins were quantified by Bradford assay (Sigma).
      Western blotting was performed and detected as described previously (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ). In brief, the primary antibodies used were as follows: rabbit polyclonal anti-EGFR (1:500, Santa Cruz Biotechnology, catalog no. SC3); mouse monoclonal anti-EGFR clone 8G6.2 (1:500, Millipore/Sigma, catalog no. 05-1047); mouse monoclonal anti-α-tubulin (1:2000, Sigma, catalog no. T6199); rabbit polyclonal anti-lamin B1 (1:2000, Abcam, catalog no. ab16048); monoclonal anti-clathrin (1:500, Abcam, catalog no. b21679); mouse monoclonal anti-PLCγ1 (1:1000, Cell Signaling Technology, catalog no. I2822); rabbit polyclonal anti-PLCδ4 (1:500, Santa Cruz Biotechnology, catalog no. H-250); rabbit polyclonal Erk1/2 (1:1000; Cell Signaling, catalog no. 9101); rabbit monoclonal anti-phospho-Erk1/2 Thr-202/Tyr-204 (1:1000; Cell Signaling, catalog no. 4370); rabbit monoclonal anti-AKT (1:1000, Cell Signaling, catalog no. 4691); rabbit monoclonal anti-phospho-AKT Thr-308 (1:1000; Cell Signaling, catalog no. 13038), cyclins A, B1, D1, D2, D3, E, E2 and H (1:500 to 1000, Cell Signaling Technology, Kit catalog no. 9869). The membranes were developed using ECL Plus (GE Healthcare). Subsequently, the films were scanned and analyzed using ImageJ software. All antibodies used were found to have the correct molecular weight by Western blotting. The antibodies for clathrin, PLCδ4, and PLCγ1 were validated with at least two specific siRNAs.

      Immunofluorescence

      Confocal and super-resolution immunofluorescence imagings were performed as described previously (
      • Faraco C.C.F.
      • Faria J.A.Q.A.
      • Kunrath-Lima M.
      • Miranda M.C.
      • de Melo M.I.A.
      • Ferreira A.D.F.
      • Rodrigues M.A.
      • Gomes D.A.
      Translocation of epidermal growth factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
      ). In brief, cells were labeled with rabbit polyclonal anti-EGFR (1:250, Santa Cruz Biotechnology, catalog no. SC3) or mouse monoclonal anti-EGFR clone 8G6.2 (1:200, Millipore/Sigma, catalog no. 05-1047) and rabbit polyclonal anti-PLCγ1 (1:200, Cell Signaling, catalog no. I2822) and polyclonal anti-PLCδ4 (Santa Cruz Biotechnology, catalog no. H-250) and then incubated with secondary antibodies conjugated to Alexa Fluor 488 or 546 (Life Technologies, Inc.). Hoechst 33342 (Life Technologies, Inc.) was used as a marker for the nuclear compartment. Images were collected using a Leica SP8 Gated STED super-resolution microscope with a ×100, 1.4 NA objective lens.

      siRNA transfection

      Cells were transfected using Lipofectamine RNAiMAX reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. 50 nm siRNAs were used for clathrin heavy chain knockdown (CHC2 siRNA 1, 5′-GCAAUUAAUUAAGGCUGACCGUAtt-3′, Life Technologies, Inc., catalog no. s223263; CHC2 siRNA 2, 5′-AGGUGGCUUCUAAAUAUCAUGAACA-3′, IDT, catalog no. hs.Ri.CLTC.13.1). 25 nm siRNAs were used for PLCγ1 knockdown (PLCγ1 siRNA 1, 5′-GAAUCGUGAGGAUCGUAUAtt-3′, Life Technologies, Inc., catalog no. s10632; and PLCγ1 siRNA 2, 5′-GACCUCAUCAGCUACUAUGAGAAAC-3′, IDT, catalog no. hs.Ri.PLCG1.13.1). For PLCδ4 knockdown, 50 nm of the following siRNAs were used: ON-TARGETplus SMARTpool (PLCδ4 siRNA 1, 5′-CAAGAAGUUCAGCGGUUAU-3′, 5′-GCUCAAUCCCAUACCGACA-3′, 5′-GACCAAUGGCUGAGCGAUU-3′, and CAACAAGGUUACCGCCACA); Dharmacon, catalog no. L-005065-01 (PLCδ4 siRNA 2, 5′-AAGGCAGGUUGCAACUAGAAAUUCA-3′) (IDT, catalog no. hs.Ri.PLCD4.13.3). 50 nm of the following nontargeting siRNAs were used: SilencerTM Select Negative Control No. 2 siRNA (control siRNA 1, Life Technologies, Inc., catalog no. 4390846) and IDT Scrambled negative control DsiRNA (control siRNA 2, 5′- CUUCCUCUCUUUCUCUCCCUUGUGA-3′, catalog no. 51-01-19-08). Further analysis was performed 48 h after transfection.

      InsP3-buffer constructs and calcium imaging

      Generation of InsP3-buffer constructs was as described previously (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ). Briefly, the InsP3-binding domain (residues 224–605) of the human type I InsP3 receptor was tagged with an mRFP and with a nuclear localization signal or nuclear exclusion signal to generate the nuclear and cytosolic constructs, respectively. The adenoviruses were built by ViraQuest Inc. Cells were infected with 10 multiplicities of infection, and analysis was performed in 48 h. For calcium imaging, cells were incubated with 5 μm Fluo-4/AM (Life Technologies, Inc.) and placed in a custom-made perfusion chamber for EGF stimulation. Images were collected with a Zeiss LSM 710 DUO NLO. Nuclear and cytosolic regions were selected with the assistance of the brightfield images.

      Nuclear PI(4,5)P2 extraction and detection

      PI(4,5)P2 from samples was detected by a mass ELISA kit (Echelon Biosciences, UT). 5 min after EGF stimulation, cell nuclei were isolated as described previously (
      • Gomes D.A.
      • Rodrigues M.A.
      • Leite M.F.
      • Gomez M.V.
      • Varnai P.
      • Balla T.
      • Bennett A.M.
      • Nathanson M.H.
      c-Met must translocate to the nucleus to initiate calcium signals.
      ,
      • Rodrigues M.A.
      • Gomes D.A.
      • Andrade V.A.
      • Leite M.F.
      • Nathanson M.H.
      Insulin induces calcium signals in the nucleus of rat hepatocytes.
      ), washed three times with PBS, and followed by PI(4,5)P2 extraction as recommended by the kit manufacturer. In summary, the protein was precipitated with 0.5 m TCA and followed by neutral lipid extraction using MeOH/CHCl3 (2:1) and acidic lipid extracted with MeOH, CHCl3, 12 m HCl (80:40:1). Lipids collected from split organic phase were dried before analysis.

      FRET-based PKC activity reporter

      The NucCKAR construct was developed by Gallegos et al. (
      • Gallegos L.L.
      • Kunkel M.T.
      • Newton A.C.
      Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
      ) and obtained from Addgene (plasmid no. 14869). Cells were transfected using FuGENE 6 (Promega), and microscope studies were performed in 48 h. 200 nm PdBu (Sigma) was used as a positive control. 250 nm Gö6983 (Tocris Bioscience, Bristol, UK) was used as a PKC inhibitor to confirm the specificity of EGF response. Pre-stimulation baseline was performed with only DMSO before perfusion with PdBu and Gö6983 as a vehicle control. Cells were placed in a custom-made perfusion chamber for agonist stimulation. CFP and YFP emission intensities were collected using a Zeiss LSM 710 DUO NLO with a ×63, 1.4 NA objective lens. With this construct, increased PKC activity results in decreased rather than increased FRET (
      • Gallegos L.L.
      • Kunkel M.T.
      • Newton A.C.
      Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
      ). Therefore, CFP/YFP rather than YFP/CFP emission ratio was calculated to reflect PKC activity (
      • Gallegos L.L.
      • Kunkel M.T.
      • Newton A.C.
      Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
      ) and was normalized using Microsoft Excel software.

      FRET-based InsP3 reporter

      The FRET-based InsP3-biosensor with nuclear localization signal (FIRE-1nuc) was developed by Kapoor et al. (
      • Kapoor N.
      • Maxwell J.T.
      • Mignery G.A.
      • Will D.
      • Blatter L.A.
      • Banach K.
      Spatially defined InsP3-mediated signaling in embryonic stem cell-derived cardiomyocytes.
      ). Cells were transfected using FuGENE 6 (Promega), and microscopic studies were performed in 48 h. Cells were placed in a custom-made perfusion chamber for agonist stimulation. CFP and YFP emission intensities were collected using a Zeiss LSM 710 DUO NLO with a ×63, 1.4 NA objective lens. YFP/CFP emission ratio was calculated and normalized using ZEN (Zeiss), Microsoft Excel, and GraphPad Prism software.

      Proliferation assays

      SKHEP-1 cells were seeded into 96-well plates (Corning, NY) at a density of 1 × 104 and transfected with Lipofectamine RNAiMAX with control siRNAs, PLCδ4 siRNAs, and PLCγ1 siRNAs. The cells were transfected with 50 nm of each siRNA. Cells without transfection reagent and with Lipofectamine were used as a control group. The Cell Proliferation ELISA kit (Roche Applies Science, offered by Sigma) was used according to the manufacturer’s instructions. Briefly, after 48 h, cells were cultivated with BrdU (0.2 μm) for 2 h in serum-free media, fixated for 30 min, and incubated with anti-BrdU–POD for 90 min at room temperature. After three washing steps, 3,3′,5,5′-Tetramethylbenzidine was added, and the plate was read at 370 and 492 nm (Synergy 2, Biotek).
      For proliferation curve assay, SKHep-1 cells were transfected with control siRNA 1 or PLCδ4 siRNA 1. After 48 h, cells were collected by trypsinization and counted in a Neubauer chamber using trypan blue for viability exclusion in triplicate.
      For the proliferation recovery experiment, the Tet-pLKO-neo plasmid (Addgene ID. 21916) was used to express shRNAs under the control of a doxycycline promoter. The PLCD4 shRNAs target sequences were obtained from “The RNAi Consortium Collection” (https://portals.broadinstitute.org/gpp/public/),
      Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
      and the shRNAs were designed and cloned according to Addgene instructions. In summary, shRNA sequences were subcloned into Tet-pLKO-neo after digestion with AgeI and EcoRI. We used the following target sequences for the shRNAs: PLCδ4 shRNA 1 (5′-AGAGCAGCGTCGAGGGATATA-3′); PLCδ4 shRNA 2 (5′-ACTACCACTTCTACGAGATAT-3′); scramble shRNA 1 (5′-CCTAAGGTTAAGTCGCCCTCG-3′); and scramble shRNA 2 (5′-CUUCCUCUCUUUCUCUCCCUUGUGA-3′). SKHep-1 cells were transfected and selected with 500 ng/ml G418. Stable cells were treated with 1 μg/ml doxycycline for 48 h and then were kept without doxycycline for another 48 h for PLCδ4 expression recovery. BrdU assay was performed as described above.

      Cell death assay

      For this experiment, the cells were incubated with 10 μg/ml doxorubicin for 24 h, as a positive control for cell death. The cells were incubated with annexin V–FITC (Life Technologies, Inc.) and propidium iodide (Life Technologies, Inc.) for 1 h. Images were collected with a ×10 and ×20 objective lens on an Olympus IX70 inverted fluorescence microscope (Olympus America, Melville, NY), and the data obtained were quantified by counting cell labeled for annexin V–FITC, propidium iodide, double-labeled, and crossed with bright-field images used to obtain total cell count. Each treatment was made in triplicate, and three different images were counted, and the means were used for statistical analysis.

      Statistical analysis

      The significance of changes in treatment groups was determined by Student’s t test or one-way analysis of variance with Tukey’s multiple comparison tests if not otherwise stated, using GraphPad Prism software. Data are represented as mean ± S.E.

      Author contributions

      M. C. d. M., M. A. R., A. C. d. A. C., J. A. Q. A. F., D. S., A. M. G., M. H. N., and D. A. G. conceptualization; M. C. d. M., M. A. R., A. C. d. A. C., J. A. Q. A. F., M. K.-L., and D. A. G. formal analysis; M. C. d. M., A. C. d. A. C., J. A. Q. A. F., M. K.-L., M. H. N., and D. A. G. investigation; M. C. d. M., A. C. d. A. C., G. A. M., M. H. N., and D. A. G. methodology; M. C. d. M., M. A. R., J. A. Q. A. F., G. A. M., D. S., A. M. G., M. H. N., and D. A. G. writing-review and editing; M. A. R., A. C. d. A. C., J. A. Q. A. F., D. S., A. M. G., M. H. N., and D. A. G. writing-original draft; M. A. R. and D. A. G. project administration; A. C. d. A. C., J. A. Q. A. F., and M. K.-L. data curation; J. A. Q. A. F. and D. A. G. supervision; J. A. Q. A. F. and M. H. N. visualization; G. A. M. and M. H. N. resources; M. H. N. and D. A. G. funding acquisition.

      Acknowledgments

      We acknowledge Dr. Maria Ciarleglio for the statistical consultation. We also thank “Centro de Aquisição e Processamento de Imagens do ICB/UFMG” (CAPI) for technical support.

      References

        • Berridge M.J.
        The inositol trisphosphate/calcium signaling pathway in health and disease.
        Physiol. Rev. 2016; 96 (27512009): 1261-1296
        • Pusl T.
        • Wu J.J.
        • Zimmerman T.L.
        • Zhang L.
        • Ehrlich B.E.
        • Berchtold M.W.
        • Hoek J.B.
        • Karpen S.J.
        • Nathanson M.H.
        • Bennett A.M.
        Epidermal growth factor-mediated activation of the ETS domain transcription factor Elk-1 requires nuclear calcium.
        J. Biol. Chem. 2002; 277 (11971908): 27517-27527
        • Rodrigues M.A.
        • Gomes D.A.
        • Leite M.F.
        • Grant W.
        • Zhang L.
        • Lam W.
        • Cheng Y.-C.
        • Bennett A.M.
        • Nathanson M.H.
        Nucleoplasmic calcium is required for cell proliferation.
        J. Biol. Chem. 2007; 282 (17420246): 17061-17068
        • Hardingham G.E.
        • Chawla S.
        • Johnson C.M.
        • Bading H.
        Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression.
        Nature. 1997; 385 (9000075): 260-265
        • Echevarría W.
        • Leite M.F.
        • Guerra M.T.
        • Zipfel W.R.
        • Nathanson M.H.
        Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum.
        Nat. Cell Biol. 2003; 5 (12717445): 440-446
        • Gomes D.A.
        • Leite M.F.
        • Bennett A.M.
        • Nathanson M.H.
        Calcium signaling in the nucleus.
        Can. J. Physiol. Pharmacol. 2006; 84 (16902580): 325-332
        • Gomes D.A.
        • Rodrigues M.A.
        • Leite M.F.
        • Gomez M.V.
        • Varnai P.
        • Balla T.
        • Bennett A.M.
        • Nathanson M.H.
        c-Met must translocate to the nucleus to initiate calcium signals.
        J. Biol. Chem. 2008; 283 (18073207): 4344-4351
        • Rodrigues M.A.
        • Gomes D.A.
        • Andrade V.A.
        • Leite M.F.
        • Nathanson M.H.
        Insulin induces calcium signals in the nucleus of rat hepatocytes.
        Hepatology. 2008; 48 (18798337): 1621-1631
        • Subramanian K.
        • Meyer T.
        Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores.
        Cell. 1997; 89 (9200614): 963-971
        • Malhas A.
        • Goulbourne C.
        • Vaux D.J.
        The nucleoplasmic reticulum: form and function.
        Trends Cell Biol. 2011; 21 (21514163): 362-373
        • Stehno-Bittel L.
        • Lückhoff A.
        • Clapham D.E.
        Calcium release from the nucleus by InsP3 receptor channels.
        Neuron. 1995; 14 (7530018): 163-167
        • Khamphaya T.
        • Chukijrungroat N.
        • Saengsirisuwan V.
        • Mitchell-Richards K.A.
        • Robert M.E.
        • Mennone A.
        • Ananthanarayanan M.
        • Nathanson M.H.
        • Weerachayaphorn J.
        Nonalcoholic fatty liver disease impairs expression of the type II inositol 1,4,5-trisphosphate receptor.
        Hepatology. 2018; 67 (29023819): 560-574
        • Ueasilamongkol P.
        • Khamphaya T.
        • Guerra M.T.
        • Rodrigues M.A.
        • Gomes D.A.
        • Kong Y.
        • Wei W.
        • Jain D.
        • Trampert D.C.
        • Ananthanarayanan M.
        • Banales J.M.
        • Roberts L.R.
        • Farshidfar F.
        • Nathanson M.H.
        • Weerachayaphorn J.
        Type 3 inositol 1,4,5-trisphosphate receptor is increased and enhances malignant properties in cholangiocarcinoma.
        Hepatology 2019. 2019; (31251815)
        • Brand T.M.
        • Iida M.
        • Li C.
        • Wheeler D.L.
        The nuclear epidermal growth factor receptor signaling network and its role in cancer.
        Discov. Med. 2011; 12 (22127113): 419-432
        • Li C.
        • Iida M.
        • Dunn E.F.
        • Ghia A.J.
        • Wheeler D.L.
        Nuclear EGFR contributes to acquired resistance to cetuximab.
        Oncogene. 2009; 28 (19684613): 3801-3813
        • Huang W.C.
        • Chen Y.J.
        • Li L.Y.
        • Wei Y.L.
        • Hsu S.C.
        • Tsai S.L.
        • Chiu P.C.
        • Huang W.P.
        • Wang Y.N.
        • Chen C.H.
        • Chang W.C.
        • Chang W.C.
        • Chen A.J.
        • Tsai C.H.
        • Hung M.C.
        Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells.
        J. Biol. Chem. 2011; 286 (21487020): 20558-20568
        • De Angelis Campos A.C.
        • Rodrigues M.A.
        • de Andrade C.
        • de Goes A.M.
        • Nathanson M.H.
        • Gomes D.A.
        Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway.
        Biochem. Biophys. Res. Commun. 2011; 412 (21821003): 341-346
        • Faraco C.C.F.
        • Faria J.A.Q.A.
        • Kunrath-Lima M.
        • Miranda M.C.
        • de Melo M.I.A.
        • Ferreira A.D.F.
        • Rodrigues M.A.
        • Gomes D.A.
        Translocation of epidermal growth factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.
        J. Struct. Biol. 2018; 202 (29277356): 61-69
        • Lin S.Y.
        • Makino K.
        • Xia W.
        • Matin A.
        • Wen Y.
        • Kwong K.Y.
        • Bourguignon L.
        • Hung M.C.
        Nuclear localization of EGF receptor and its potential new role as a transcription factor.
        Nat. Cell Biol. 2001; 3 (11533659): 802-808
        • Huo L.
        • Wang Y.N.
        • Xia W.
        • Hsu S.C.
        • Lai C.C.
        • Li L.Y.
        • Chang W.C.
        • Wang Y.
        • Hsu M.C.
        • Yu Y.L.
        • Huang T.H.
        • Ding Q.
        • Chen C.H.
        • Tsai C.H.
        • Hung M.C.
        RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (20802156): 16125-16130
        • Wang S.C.
        • Nakajima Y.
        • Yu Y.L.
        • Xia W.
        • Chen C.T.
        • Yang C.C.
        • McIntush E.W.
        • Li L.Y.
        • Hawke D.H.
        • Kobayashi R.
        • Hung M.C.
        Tyrosine phosphorylation controls PCNA function through protein stability.
        Nat. Cell Biol. 2006; 8 (17115032): 1359-1368
        • Liccardi G.
        • Hartley J.A.
        • Hochhauser D.
        EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment.
        Cancer Res. 2011; 71 (21266349): 1103-1114
        • Wang Y.N.
        • Hung M.C.
        Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family.
        Cell Biosci. 2012; 2 (22520625): 13
        • Bootman M.D.
        • Fearnley C.
        • Smyrnias I.
        • MacDonald F.
        • Roderick H.L.
        An update on nuclear calcium signalling.
        J. Cell Sci. 2009; 122 (19571113): 2337-2350
        • Nakamura Y.
        • Fukami K.
        Regulation and physiological functions of mammalian phospholipase C.
        J. Biochem. 2017; 161 (28130414): 315-321
        • Faenza I.
        • Fiume R.
        • Piazzi M.
        • Colantoni A.
        • Cocco L.
        Nuclear inositide specific phospholipase C signalling–interactions and activity.
        FEBS J. 2013; 280 (23890371): 6311-6321
        • Leung D.W.
        • Tompkins C.
        • Brewer J.
        • Ball A.
        • Coon M.
        • Morris V.
        • Waggoner D.
        • Singer J.W.
        Phospholipase C δ-4 overexpression upregulates ErB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells.
        Mol. Cancer. 2004; 3 (15140260): 15
        • Ratti S.
        • Mongiorgi S.
        • Ramazzotti G.
        • Follo M.Y.
        • Mariani G.A.
        • Suh P.G.
        • McCubrey J.A.
        • Cocco L.
        • Manzoli L.
        Nuclear inositide signaling via phospholipase C.
        J. Cell Biochem. 2017; 118 (28106288): 1969-1978
        • Lee S.B.
        • Rhee S.G.
        Molecular cloning, splice variants, expression, and purification of phospholipase C-δ4.
        J. Biol. Chem. 1996; 271 (8550568): 25-31
        • Nagano K.
        • Fukami K.
        • Minagawa T.
        • Watanabe Y.
        • Ozaki C.
        • Takenawa T.
        A novel phospholipase C δ4 (PLCδ4) splice variant as a negative regulator of PLC.
        J. Biol. Chem. 1999; 274 (9915823): 2872-2879
        • Kunrath-Lima M.
        • de Miranda M.C.
        • Ferreira A.D.F.
        • Faraco C.C.F.
        • de Melo M.I.A.
        • Goes A.M.
        • Rodrigues M.A.
        • Faria J.A.Q.A.
        • Gomes D.A.
        Phospholipase Cδ4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.
        Cell. Signal. 2018; 49 (29859928): 59-67
        • Asano M.
        • Tamiya-Koizumi K.
        • Homma Y.
        • Takenawa T.
        • Nimura Y.
        • Kojima K.
        • Yoshida S.
        Purification and characterization of nuclear phospholipase C specific for phosphoinositides.
        J. Biol. Chem. 1994; 269 (8163540): 12360-12366
        • Liu N.
        • Fukami K.
        • Yu H.
        • Takenawa T.
        A new phospholipase Cδ4 is induced at S-phase of the cell cycle and appears in the nucleus.
        J. Biol. Chem. 1996; 271 (8550586): 355-360
        • Moccia F.
        • Berra-Romani R.
        • Tritto S.
        • Signorelli S.
        • Taglietti V.
        • Tanzi F.
        Epidermal growth factor induces intracellular Ca2+ oscillations in microvascular endothelial cells.
        J. Cell Physiol. 2003; 194 (12494452): 139-150
        • Martelli A.M.
        • Evangelisti C.
        • Nyakern M.
        • Manzoli F.A.
        Nuclear protein kinase C.
        Biochim. Biophys. Acta. 2006; 1761 (16574477): 542-551
        • Gallegos L.L.
        • Kunkel M.T.
        • Newton A.C.
        Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling.
        J. Biol. Chem. 2006; 281 (16901905): 30947-30956
        • Shi Y.
        • Tao Y.
        • Jiang Y.
        • Xu Y.
        • Yan B.
        • Chen X.
        • Xiao L.
        • Cao Y.
        Nuclear epidermal growth factor receptor interacts with transcriptional intermediary factor 2 to activate cyclin D1 gene expression triggered by the oncoprotein latent membrane protein 1.
        Carcinogenesis. 2012; 33 (22581837): 1468-1478
        • Sandvig K.
        • van Deurs B.
        Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin.
        FEBS Lett. 2002; 529 (12354612): 49-53
        • Marsh M.
        • Helenius A.
        Virus entry: open sesame.
        Cell. 2006; 124 (16497584): 729-740
        • Carpenter G.
        • Liao H.J.
        Trafficking of receptor tyrosine kinases to the nucleus.
        Exp. Cell Res. 2009; 315 (18951890): 1556-1566
        • Follo M.Y.
        • Finelli C.
        • Mongiorgi S.
        • Clissa C.
        • Bosi C.
        • Testoni N.
        • Chiarini F.
        • Ramazzotti G.
        • Baccarani M.
        • Martelli A.M.
        • Manzoli L.
        • Martinelli G.
        • Cocco L.
        Reduction of phosphoinositide-phospholipase C β1 methylation predicts the responsiveness to azacitidine in high-risk MDS.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106 (19805378): 16811-16816
        • Nishibe S.
        • Wahl M.I.
        • Hernández-Sotomayor S.M.
        • Tonks N.K.
        • Rhee S.G.
        • Carpenter G.
        Increase of the catalytic activity of phospholipase C-γ1 by tyrosine phosphorylation.
        Science. 1990; 250 (1700866): 1253-1256
        • Kumar V.
        • Jong Y.J.
        • O'Malley K.L.
        Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release.
        J. Biol. Chem. 2008; 283 (18337251): 14072-14083
        • Manzoli L.
        • Billi A.M.
        • Rubbini S.
        • Bavelloni A.
        • Faenza I.
        • Gilmour R.S.
        • Rhee S.G.
        • Cocco L.
        Essential role for nuclear phospholipase Cβ1 in insulin-like growth factor I-induced mitogenesis.
        Cancer Res. 1997; 57 (9187110): 2137-2139
        • Faenza I.
        • Ramazzotti G.
        • Bavelloni A.
        • Fiume R.
        • Gaboardi G.C.
        • Follo M.Y.
        • Gilmour R.S.
        • Martelli A.M.
        • Ravid K.
        • Cocco L.
        Inositide-dependent phospholipase C signaling mimics insulin in skeletal muscle differentiation by affecting specific regions of the cyclin D3 promoter.
        Endocrinology. 2007; 148 (17122077): 1108-1117
        • Ramazzotti G.
        • Faenza I.
        • Gaboardi G.C.
        • Piazzi M.
        • Bavelloni A.
        • Fiume R.
        • Manzoli L.
        • Martelli A.M.
        • Cocco L.
        Catalytic activity of nuclear PLC-β1 is required for its signalling function during C2C12 differentiation.
        Cell. Signal. 2008; 20 (18694821): 2013-2021
        • Huang W.
        • Mishra V.
        • Batra S.
        • Dillon I.
        • Mehta K.D.
        Phorbol ester promotes histone H3-Ser10 phosphorylation at the LDL receptor promoter in a protein kinase C-dependent manner.
        J. Lipid Res. 2004; 45 (15145978): 1519-1527
        • Parekh D.B.
        • Ziegler W.
        • Parker P.J.
        Multiple pathways control protein kinase C phosphorylation.
        EMBO J. 2000; 19 (10675318): 496-503
        • Fima E.
        • Shtutman M.
        • Libros P.
        • Missel A.
        • Shahaf G.
        • Kahana G.
        • Livneh E.
        PKCη enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.
        Oncogene. 2001; 20 (11709714): 6794-6804
        • Poli A.
        • Faenza I.
        • Chiarini F.
        • Matteucci A.
        • McCubrey J.A.
        • Cocco L.
        K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway.
        Cell Cycle. 2013; 12 (23656785): 1713-1721
        • Poli A.
        • Ramazzotti G.
        • Matteucci A.
        • Manzoli L.
        • Lonetti A.
        • Suh P.G.
        • McCubrey J.A.
        • Cocco L.
        A novel DAG-dependent mechanism links PKCα and cyclin B1 regulating cell cycle progression.
        Oncotarget. 2014; 5 (25362646): 11526-11540
        • Fiume R.
        • Ramazzotti G.
        • Teti G.
        • Chiarini F.
        • Faenza I.
        • Mazzotti G.
        • Billi A.M.
        • Cocco L.
        Involvement of nuclear PLCβ1 in lamin B1 phosphorylation and G2/M cell cycle progression.
        FASEB J. 2009; 23 (19028838): 957-966
        • Michalopoulos G.K.
        Hepatostat: liver regeneration and normal liver tissue maintenance.
        Hepatology. 2017; 65 (27997988): 1384-1392
        • Kapoor N.
        • Maxwell J.T.
        • Mignery G.A.
        • Will D.
        • Blatter L.A.
        • Banach K.
        Spatially defined InsP3-mediated signaling in embryonic stem cell-derived cardiomyocytes.
        PLoS ONE. 2014; 9 (24409283): e83715
        • Akutagawa A.
        • Fukami K.
        • Banno Y.
        • Takenawa T.
        • Kannagi R.
        • Yokoyama Y.
        • Oda K.
        • Nagino M.
        • Nimura Y.
        • Yoshida S.
        • Tamiya-Koizumi K.
        Disruption of phospholipase Cδ4 gene modulates the liver regeneration in cooperation with nuclear protein kinase C.
        J. Biochem. 2006; 140 (16998201): 619-625
        • Boyer J.L.
        • Phillips J.M.
        • Graf J.
        Preparation and specific applications of isolated hepatocyte couplets.
        Methods Enzymol. 1990; 192 (1963665): 501-516