Intercellular Variation in Signaling through the TGF-β Pathway and Its Relation to Cell Density and Cell Cycle Phase

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment. Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment. Transforming growth factor-β (TGF-β) 1The abbreviations used are:TGF-βtransforming growth factor-βPLAproximity ligation assayRCArolling circle amplificationR-Smadreceptor regulated SmadSmadsmall mothers against decapentaplegicTβRITGF-β receptor type ITβRIITGF-β receptor type II. 1The abbreviations used are:TGF-βtransforming growth factor-βPLAproximity ligation assayRCArolling circle amplificationR-Smadreceptor regulated SmadSmadsmall mothers against decapentaplegicTβRITGF-β receptor type ITβRIITGF-β receptor type II. controls a diverse array of cellular processes, including cell proliferation, differentiation, apoptosis, and determination of developmental fate during embryogenesis (1Massagué J. Blain S.W. Lo R.S. TGFβ signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2069) Google Scholar, 2Moustakas A. Heldin C.H. The regulation of TGFβ signal transduction.Development. 2009; 136: 3699-3714Crossref PubMed Scopus (684) Google Scholar). TGF-β binding to the serine/threonine kinase type II receptor (TβRII) promotes the formation of a complex with the type I receptor (TβRI), whereafter the latter is phosphorylated and activated. Important substrates for the TβRI are the receptor-regulated Smads (R-Smads), Smad2 and Smad3 (3Massagué J. Wotton D. Transcriptional control by the TGF-β/Smad signaling system.EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar), which after C-terminal phosphorylation accumulate in the nucleus, where they form heteromeric complexes with transcriptional factors, co-repressors, and co-activators to up- or down-regulate transcription of target genes (1Massagué J. Blain S.W. Lo R.S. TGFβ signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2069) Google Scholar, 2Moustakas A. Heldin C.H. The regulation of TGFβ signal transduction.Development. 2009; 136: 3699-3714Crossref PubMed Scopus (684) Google Scholar, 4Abdollah S. Macías-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. TβRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling.J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Among the crucial limiting regulators of the TGF-β pathway are E3 ubiquitin ligases that influence the duration of Smad signaling by promoting ubiquitin-mediated proteasomal degradation of receptors and Smads. E3 ligases also promote signaling by degrading repressors of the pathway. Common mediator Smad and R-Smads form complexes with SnoN (Ski-related novel protein N) and Ski (Sloan-Kettering avian retrovirus transforming protein), transcription repressors that inhibit formation of transcriptionally active heteromeric Smad complexes or recruit co-repressor complexes to the chromatin of target genes (5Akiyoshi S. Inoue H. Hanai J. Kusanagi K. Nemoto N. Miyazono K. Kawabata M. c-Ski acts as a transcriptional co-repressor in transforming growth factor-β signaling through interaction with smads.J. Biol. Chem. 1999; 274: 35269-35277Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 6Sun Y. Liu X. Ng-Eaton E. Lodish H.F. Weinberg R.A. SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor β signaling.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12442-12447Crossref PubMed Scopus (226) Google Scholar, 7Xu W. Angelis K. Danielpour D. Haddad M.M. Bischof O. Campisi J. Stavnezer E. Medrano E.E. Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5924-5929Crossref PubMed Scopus (180) Google Scholar). SnoN ubiquitination and proteasomal degradation is a required step in activation of TGF-β signaling. Thus, in response to TGF-β, SnoN in complex with activated Smad2/3 recruits E3 ligases, which mediate its ubiquitin-dependent degradation (8Bonni S. Wang H.R. Causing C.G. Kavsak P. Stroschein S.L. Luo K. Wrana J.L. TGF-β induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation.Nat Cell Biol. 2001; 3: 587-595Crossref PubMed Scopus (272) Google Scholar, 9Stroschein S.L. Bonni S. Wrana J.L. Luo K. Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN.Genes Dev. 2001; 15: 2822-2836Crossref PubMed Google Scholar). Earlier studies of Smad interactors have mostly relied on engineered systems of transfected overexpressing cells, with measurements made across populations of cells. Because of the limitations of such methods, important questions remain about mechanisms and kinetics of endogenous cell signaling, about the localization of complexes within different cells and compartments of the cell, and about the quantitative nature of these processes. transforming growth factor-β proximity ligation assay rolling circle amplification receptor regulated Smad small mothers against decapentaplegic TGF-β receptor type I TGF-β receptor type II. transforming growth factor-β proximity ligation assay rolling circle amplification receptor regulated Smad small mothers against decapentaplegic TGF-β receptor type I TGF-β receptor type II. In this paper, we describe spatial and temporal aspects of the formation of Smad complexes in situ, under physiological conditions, using the in situ proximity ligation assay (PLA) (10Söderberg O. Gullberg M. Jarvius M. Ridderstråle K. Leuchowius K.J. Jarvius J. Wester K. Hydbring P. Bahram F. Larsson L.G. Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation.Nat. Methods. 2006; 3: 995-1000Crossref PubMed Scopus (1754) Google Scholar). The ability to resolve and enumerate individual protein-protein interaction events has enabled us to present quantitative data of Smad complex formation and localization within compartments of single cells. Our data support and extend earlier findings about TGF-β signaling and demonstrate the potential of the in situ PLA method to reveal new mechanisms of regulation of cell signaling in genetically unmodified cells and in human tissue samples at cellular and subcellular resolution. Mouse embryonic fibroblasts, a human immortalized nontransformed keratinocyte epithelial cell line (HaCaT), and a mouse mammary gland cell line (NMuMG) were grown in high glucose Dulbecco's modified Eagle's medium (Sigma). Human hepatocellular liver carcinoma (HepG2) and human breast carcinoma (MDA-MB-468) cell lines were cultured in RPMI (Sigma). Media were supplemented with 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Sigma). For in situ PLA experiments, the cells were seeded at a density of 10,000 cells/well onto SuperFrost Ultra Plus slides (Menzel Glaser) 48 h before treatment. For purposes of blocking basal TGF-β signaling in unstimulated cells, the low molecular weight inhibitor GW6604 (11Gellibert F. de Gouville A.C. Woolven J. Mathews N. Nguyen V.L. Bertho-Ruault C. Patikis A. Grygielko E.T. Laping N.J. Huet S. Discovery of 4-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide (GW788388): A potent, selective, and orally active transforming growth factor-β type I receptor inhibitor.J. Med. Chem. 2006; 49: 2210-2221Crossref PubMed Scopus (89) Google Scholar) was added to the cells at a concentration of 5 μm 2 h prior to stimulation. After washing in PBS (137 mmol/liter NaCl, 10 mmol/liter phosphate, 2.7 mmol/liter KCl, pH 7.4), the stimulated cells were incubated in the presence or absence of TGF-β1 (10 ng/ml; BioSource/Invitrogen) in Dulbecco's modified Eagle's medium for 45 min. After stimulation, the cells were fixed in 3% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature and permeabilized for 10 min with 0.5% Triton X-100 (Sigma-Aldrich) in PBS. Tissue microarrays with 4-μm cores were prepared from normal colon tissue fixed in PBS-buffered 10% formalin and embedded in paraffin according to standard pathology laboratory protocols. The tissue microarrays were then baked for 30 min at 60 °C, de-paraffinized in xylene (Histolab), and incubated at 125 °C, under high pressure conditions in a pressure cooker (Biocare Medical) in 600 ml of antigen retrieval solution (Dako) for 1 min. A thiol-modified rolling circle amplification (RCA) primer oligonucleotide SH-AAAAAAAAAATATTGACAGAACTAGACACTT or the nonpriming oligonucleotides SH-AAAAAAAAAAATGGCCGACTCACGAATTAGA[UUU] and SH-AAAAAAAAAAGACGCTAATAGTTAAGACGCTT[UUU] ([UUU] is 2′O-methyl-RNA) (Eurogentec) were used for conjugation to antibodies against Smad2, Smad3 (made at the Ludwig Institute for and the antibodies were using protein and against PBS using a were modified by with a of prepared in for 2 h at room In of oligonucleotide was with 10 in and for 1 h at The were by and to 5 in PBS using The modified antibodies and oligonucleotides were then in and against PBS at for conjugation to PLA fixed cells and tissue were incubated with blocking solution for 2 h at room temperature by with antibodies Smad2, SnoN Ski mouse or PLA complex in The was by in for 5 min in with NaCl, pH with for cells incubated with with 2 h of with and PLA in The slides were then for 5 min with The cells and the tissue were incubated with ligation solution at for 30 by in for 2 min in For of complexes, oligonucleotides and at a concentration of in ligase NaCl, and units/ml ligase in were For amplification of the ligation and of complexes, amplification solution was on slides for min at °C, by in the localization of in situ PLA in and the cells were with and the in situ the cells and the were in and incubated with or antibodies at for 30 in and with medium The were using a with a and The was used for from were and cell with of the and the For and from cells were cells were for 2 h with GW6604 inhibitor in and stimulated with TGF-β1 (10 After stimulation, the cells were in pH with 5 NaCl, and (all from were according to standard with 10% in for 1 h at room temperature as blocking and The antibodies used were and mouse The and were from and used at a in is that TGF-β signaling phosphorylation by the form complexes with and accumulate in the to regulate transcription of target complex formation endogenous Smad proteins in individual cells during TGF-β signaling, we used the in situ PLA method We Smad2-Smad4 and complex formation in mouse embryonic cells. of of complexes, by were in cells with a at 45 min after TGF-β The of complexes were in the complexes were also or quantitative were Smad2-Smad4 and complexes, the open that at a of native complexes are in Smad2-Smad3-Smad4 as by in studies A. S. M. K. basis of heteromeric protein assembly in TGF-β 15: Full Text Full Text PDF PubMed Scopus Google Scholar). we investigated complexes Smad2, and proteins in mouse embryonic cells, using a modified in situ PLA where on binding by a of against of the proteins in the The assay revealed complexes Smad2, and these complexes were in the of stimulated cells, with in the The method us to the Smad complexes in a cell or cells accumulate populations of Smad of diverse We the formation of complexes Smad3 and in a of cell lines to the of this for cellular to in and cells, we of complexes in of stimulated cells, with cells Transforming growth factor of the and Biol. 2000; PubMed Scopus Google Scholar). The of cell lines the of of Smad complexes in cells with active TGF-β signaling. formation of Smad complexes on the concentration of TGF-β added to the cells were for 45 min with TGF-β at from to and we used in situ PLA to assembly of complexes Smad2-Smad4 and and We a of in of stimulated cells using as as of the The of complexes Smad2 and was at a concentration of the assembly of complexes a at a concentration of We that TGF-β formation of Smad complexes at low in a We analyzed the kinetics of assembly of Smad2-Smad4 and complexes in cells TGF-β We of Smad2-Smad4 and complexes in of stimulated cells after min was to 45 min after the of TGF-β, the of complexes a before with intercellular variability of of complexes, the nature of a cell in of cell cycle or the of individual cells in the in of of cells and developed kinetics of assembly was for Smad2-Smad4 and complexes and The complexes at min of TGF-β active Smad complexes in the a of cell density on Smad complex we complexes of Smad3 and in cells seeded at different and cultured for h before with We investigated cells grown at high density medium density and low density and variation in of complexes and In cells grown at the the of complexes was with cells seeded and in complexes were We the in situ PLA data with the phosphorylation of as analyzed by In to the of PLA we of Smad3 phosphorylation among cells seeded at different that cell density complex formation of C-terminal also the that the of as of TGF-β signaling in cells and to signaling, the of as used Smad complexes are to ubiquitin-mediated proteasomal For this we the on Smad protein interactions by degradation using the proteasomal inhibitor in the presence of the of complexes was during the of TGF-β and cells with the TβRI inhibitor GW6604 stimulated with TGF-β also of Smad complexes and findings that in the absence of TGF-β stimulation, complexes are at low and degraded by and that blocking of this degradation to their Our quantitative data on Smad interactions in situ were with the C-terminal phosphorylation of as analyzed by and of Smad3 was after 5 min of TGF-β in the presence of and at 45 min. In in cells that phosphorylated Smad3 was at with the data for individual cells in is also that in situ PLA revealed complexes at of signaling under the physiological conditions, the Smad complexes accumulate with the kinetics We then investigated mechanisms that the of individual proteins under normal conditions, using in situ PLA to interactions of R-Smads and SnoN or Ski in cells. SnoN and Ski are to to R-Smads and to then proteasomal degradation E. Wang L. A. D. A. L. Luo K. Transforming growth factor-β the ability of Ski to inhibit by its PubMed Scopus Google Scholar, L. M. D. S. transcription by SnoN Biol. PubMed Scopus Google Scholar, K. D. the TGF-β PubMed Scopus Google Scholar). of and complexes were in cells where TβRI signaling was by GW6604 and with TGF-β a in complexes and In the presence of the of complexes with of TGF-β and at with a of the in Ski from the degradation of Ski complexes that with formation of Smad complexes. that the of rapidly Smad complexes regulated by the of the Smad SnoN and the in of complexes among individual cells in a we the that the assembly of protein complexes during the cell a for we used against a phosphorylated form of to cells from cells in We also analysis of complex formation in cells by the and by this different cell cycle The analysis revealed that cells complexes Smad proteins, cells in to TGF-β by Smad protein complexes A and and We the that the investigated Smad complexes by SnoN and Ski during these proteins are to transcriptional repressors of TGF-β signaling K. Ski and regulators of TGF-β Dev. PubMed Scopus Google Scholar). in situ PLA with antibodies against and R-Smads revealed of these complexes in cells and and and a low of complexes in cells in we found that in cells, complexes of SnoN or Ski and R-Smads were and their on cell density and on the proximity of individual cells to the of the We that of Smad complexes is during and the in limiting the of such complexes cells We investigated the of using in situ PLA to the presence of Smad proteins for purposes in the was on the of the of TGF-β signaling during normal tissue and colon (1Massagué J. Blain S.W. Lo R.S. TGFβ signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2069) Google Scholar, 2Moustakas A. Heldin C.H. The regulation of TGFβ signal transduction.Development. 2009; 136: 3699-3714Crossref PubMed Scopus (684) Google Scholar). The data Smad2-Smad4 and complexes in the of cells in normal colon A and In in as and the were in and The distribution of through the normal tissue was and to in samples from the complexes were which is with the active TGF-β signaling in or (1Massagué J. Blain S.W. Lo R.S. TGFβ signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2069) Google Scholar, 2Moustakas A. Heldin C.H. The regulation of TGFβ signal transduction.Development. 2009; 136: 3699-3714Crossref PubMed Scopus (684) Google Scholar). is that that the analyzed normal tissue were at a where TGF-β signaling was or at the where the Smad complexes also in the the normal samples that we have from different the presence of a that the Smad complexes in the under normal conditions. are investigated of cellular are at the of and of cells to in the and we have used the in situ PLA to by proteins to TGF-β at cellular and subcellular in genetically unmodified cells. We that the new cell and a basis for studies of of cellular in and in response to is that TGF-β assembly of heteromeric complexes Smad2 and or Smad3 and A. T. S. Kawabata M. A. E. K. Hanai J. Heldin C.H. Miyazono K. P. TGF-β through Smad2, Smad3 and J. 1997; PubMed Scopus Google Scholar). Smad complexes are analyzed in cells transfected with and they have in single cells. we demonstrate a to new in the signaling pathway by individual endogenous complexes activated by TGF-β at cellular and subcellular resolution. in situ we demonstrate of heteromeric complexes of R-Smads and in cell in response to TGF-β stimulation. we for the in situ the formation and of endogenous complexes of Smad2, and is in with in that the Smad complex is a A. S. M. K. basis of heteromeric protein assembly in TGF-β 15: Full Text Full Text PDF PubMed Scopus Google Scholar). The for formation are among the and it for to in a The formation of diverse of Smad complexes of different a potential of variability of by Smad The of by Smad binding of to that different of Smad The assembly of Smad complexes the of signaling complexes and to the of by trimeric complexes were in the we are to that also The quantitative in situ revealed of cells with different to TGF-β as by of protein complexes. We that the of complexes on the cellular environment and the density of the cells also as a of the of the cell cycle and and The ability of TGF-β to assembly of Smad protein complexes was in cell cells at the of or in small of cells of interactions we in the phosphorylation of the Smad3 in cells. Smad3 has to phosphorylated by TGF-β receptors at its also by in the which the formation of complexes with Smads and the transcriptional Wang D. J. Liu F. regulate the of PubMed Scopus Google Scholar, F. of Smad by PubMed Scopus Google Scholar). Smad3 to of phosphorylated at its has also that proteins, such as proteins that activated by the pathway are to the phosphorylated of Smad proteins in a cell in of phosphorylated Smads and of TGF-β signaling X. P. M. A. K. J. Wrana J.L. The complex cell density to control of the 19: Full Text Full Text PDF PubMed Scopus Google Scholar). the TGF-β signaling pathway by a of such as the dynamics of Smad phosphorylation in response to diverse the presence of and the cell M. A. C. F. J. S. M. phosphorylation during 3: PubMed Scopus Google Scholar). We used phosphorylated as a with PLA analysis of Smad protein interactions on cells, that the of complexes of and R-Smads were in cells a cell regulation of TGF-β signaling and is to that in where TGF-β induces cell cycle at the (1Massagué J. Blain S.W. Lo R.S. TGFβ signaling in growth control, cancer, and heritable disorders.Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2069) Google Scholar, 2Moustakas A. Heldin C.H. The regulation of TGFβ signal transduction.Development. 2009; 136: 3699-3714Crossref PubMed Scopus (684) Google Scholar), the formation of Smad complexes active at this cell cycle phase. that TGF-β signaling with the physiological this in the epithelial cells. Our findings are also with from where Smad proteins were to during and degraded by in E. D. of proteins for Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that the signaling Smad proteins during is of Smads by SnoN and which regulate the of signaling and to TGF-β J. A. Luo K. SnoN in normal and cells TGF-β signaling by of the Smad Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Earlier studies have that SnoN and Ski proteins are regulated in a cell manner M. Y. Wang W. E. Liu X. of cell degradation of c-Ski by PubMed Scopus Google Scholar, Y. Liu X. The anaphase-promoting complex TGF-β signaling by SnoN for 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is also that Smad3 phosphorylation at the or inhibit transcriptional of Smad3 and ubiquitination and proteasomal degradation by Ski and on during the human cell cycle have that protein phosphorylation in are and phosphorylated in cells M. A. C. F. J. S. M. phosphorylation during 3: PubMed Scopus Google Scholar). these proteins to We found that the of complexes and R-Smads in a manner in cells high of that SnoN and Ski Smad proteins and regulate transcription during mitosis. the in of Ski and complexes with TGF-β, which by and the localization of these complexes, that the ability of Ski to R-Smads on that SnoN and Ski with Smad complex formation in the absence of TGF-β stimulation, a low of signaling. We also report for the the of Smad complexes in normal and human The of TGF-β signaling in the normal or In studies of TGF-β signaling in the colon to of or pathological such as E. E. H. in and Cell Dev. Biol. PubMed Scopus Google Scholar). We are in the of TGF-β signaling using in situ PLA in colon and normal during mouse and we also the distribution of Smad protein complexes in from with colon M. O. U. O. in The of the cell that Smad complexes is that we at The provided on the of phosphorylated R-Smads and complexes that in situ PLA have for analysis of active TGF-β signaling in human providing a for this important signaling pathway. Our demonstrate that the ability to Smad interactions at the of individual cells crucial information about cell signaling and its dependence on cell cycle and density of cell populations N. A. N. U. of the in individual PubMed Scopus Google Scholar, D. of PubMed Scopus Google Scholar, S. T. dynamics reveal activation and information PubMed Scopus Google Scholar). We the to important cell signaling events in a of cell and in tissue Thus, analysis of Smad proteins in situ PLA new for developmental and studies of signaling with