| 1. |
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| 2. |
- Hammond, Maria, et al.
(författare)
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Profiling cellular protein complexes by proximity ligation with dual tag microarray readout
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:7, s. e40405-
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Tidskriftsartikel (refereegranskat)abstract
- Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.
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| 3. |
- Hochdorfer, Thomas, et al.
(författare)
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Expression of c-Kit discriminates between two functionally distinct subsets of human type 2 innate lymphoid cells
- 2019
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Ingår i: European Journal of Immunology. - : WILEY. - 0014-2980 .- 1521-4141. ; 49:6, s. 884-893
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Tidskriftsartikel (refereegranskat)abstract
- Human type 2 innate lymphoid cells (ILC2) are the only ILC subset that shows heterogeneous expression of the SCF receptor c-Kit (CD117). Despite its use as surface marker to distinguish ILC populations, its influence on ILC2 biology has not been investigated. Here, we show that c-Kit expression of peripheral blood ILC distinguishes two functionally distinct ILC2 subsets (c-Kit(hi) and c-Kit(lo)). When examined for their potential for functional plasticity we found that c-Kit(lo) ILC2 displayed greater potential to produce type 2 cytokines, possibly representing fully mature and lineage committed ILC2. On the other hand, c-Kit(hi) ILC2 coexpressed the ILC3-marker and chemokine receptor CCR6 and were able to mount a significant IL-17A response under ILC3-promoting conditions. In addition, c-Kit(hi) ILC2 produced higher levels of IFN-gamma than c-Kit(lo) ILC2 under ILC1-conditions. Although costimulation with SCF did not further influence ILC2 plasticity, it augmented type 2 cytokine production. We conclude that c-Kit marks distinct subpopulations of ILC2, which has therapeutic implications for conditions in which ILC2 are involved, such as allergy and asthma.
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| 4. |
- Kurisaki, Keiko, et al.
(författare)
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Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation
- 2003
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 23:13, s. 4494-4510
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Tidskriftsartikel (refereegranskat)abstract
- Smad proteins transduce transforming growth factor beta (TGF-beta) and bone morphogenetic protein (BMP) signals that regulate cell growth and differentiation. We have identified YY1, a transcription factor that positively or negatively regulates transcription of many genes, as a novel Smad-interacting protein. YY1 represses the induction of immediate-early genes to TGF-beta and BMP, such as the plasminogen activator inhibitor 1 gene (PAI-1) and the inhibitor of differentiation/inhibitor of DNA binding 1 gene (Id-1). YY1 inhibits binding of Smads to their cognate DNA elements in vitro and blocks Smad recruitment to the Smad-binding element-rich region of the PAI-1 promoter in vivo. YY1 interacts with the conserved N-terminal Mad homology 1 domain of Smad4 and to a lesser extent with Smad1, Smad2, and Smad3. The YY1 zinc finger domain mediates the association with Smads and is necessary for the repressive effect of YY1 on Smad transcriptional activity. Moreover, downregulation of endogenous YY1 by antisense and small interfering RNA strategies results in enhanced transcriptional responses to TGF-beta or BMP. Ectopic expression of YY1 inhibits, while knockdown of endogenous YY1 enhances, TGF-beta- and BMP-induced cell differentiation. In contrast, overexpression or knockdown of YY1 does not affect growth inhibition induced by TGF-beta or BMP. Accordingly, YY1 does not interfere with the regulation of immediate-early genes involved in the TGF-beta growth-inhibitory response, the cell cycle inhibitors p15 and p21, and the proto-oncogene c-myc. In conclusion, YY1 represses Smad transcriptional activities in a gene-specific manner and thus regulates cell differentiation induced by TGF-beta superfamily pathways.
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| 5. |
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| 6. |
- Moustakas, Aristidis, et al.
(författare)
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Mechanisms of TGF-beta signaling in regulation of cell growth and differentiation
- 2002
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Ingår i: Immunology Letters. - 0165-2478 .- 1879-0542. ; 82:1-2, s. 85-91
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Forskningsöversikt (refereegranskat)abstract
- Transforming growth factor beta (TGF-beta) is a secreted protein that regulates proliferation, differentiation and death of various cell types. All immune cell lineages, including B, T and dendritic cells as well as macrophages, secrete TGF-beta, which negatively regulates their proliferation, differentiation and activation by other cytokines. Thus, TGF-beta is a potent immunosuppressor and perturbation of TGF-beta signaling is linked to autoimmunity, inflammation and cancer. Regulation of cell proliferation and differentiation by TGF-beta is a topic of great basic and clinical importance. We summarize our work on the growth inhibitory pathway downstream of TGF-beta, which is triggered by receptor serine/threonine kinases at the cell surface and downstream effectors of the Smad family. Activated Smads regulate transcription of target genes, including cell cycle inhibitors such as p21, which mediate the anti-proliferative response and partially explain the tumor suppressive action of the TGF-beta pathway. We have described a molecular mechanism of regulation of the p21 gene by Smads and transcription factor Sp1. At late stages of tumor progression, TGF-beta promotes tumorigenesis via suppression of the immune system and changes in cell differentiation of epithelial tumor cells, a phenomenon termed epithelial to mesenchymal transdifferentiation (EMT). We review our work on the role of the Smad pathway in controlling EMT. In conclusion, the molecular pathways that describe the anti-proliferative and transdifferentiating effects of TGF-beta in epithelial cells have been uncovered to great molecular detail; a future challenge will be to test their generality in other systems, including the immune system.
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| 7. |
- Niimi, Hideki, et al.
(författare)
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Notch signaling is necessary for epithelial growth arrest by TGF-beta
- 2007
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 176:5, s. 695-707
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor beta (TGF-beta) and Notch act as tumor suppressors by inhibiting epithelial cell proliferation. TGF-beta additionally promotes tumor invasiveness and metastasis, whereas Notch supports oncogenic growth. We demonstrate that TGF-beta and ectopic Notch1 receptor cooperatively arrest epithelial growth, whereas endogenous Notch signaling was found to be required for TGF-beta to elicit cytostasis. Transcriptomic analysis after blocking endogenous Notch signaling uncovered several genes, including Notch pathway components and cell cycle and apoptosis factors, whose regulation by TGF-beta requires an active Notch pathway. A prominent gene coregulated by the two pathways is the cell cycle inhibitor p21. Both transcriptional induction of the Notch ligand Jagged1 by TGF-beta and endogenous levels of the Notch effector CSL contribute to p21 induction and epithelial cytostasis. Cooperative inhibition of cell proliferation by TGF-beta and Notch is lost in human mammary cells in which the p21 gene has been knocked out. We establish an intimate involvement of Notch signaling in the epithelial cytostatic response to TGF-beta.
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| 8. |
- Pardali, Katerina, et al.
(författare)
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Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer
- 2007
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1775:1, s. 21-62
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Forskningsöversikt (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) is a secreted polypeptide that signals via receptor serine/threonine kinases and intracellular Smad effectors. TGF-beta inhibits proliferation and induces apoptosis in various cell types, and accumulation of loss-of-function mutations in the TGF-beta receptor or Smad genes classify the pathway as a tumor suppressor in humans. In addition, various oncogenic pathways directly inactivate the TGF-beta receptor-Smad pathway, thus favoring tumor growth. On the other hand, all human tumors overproduce TGF-beta whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Accordingly, TGF-beta induces epithelial-mesenchymal transition, a differentiation switch that is required for transitory invasiveness of carcinoma cells. Tumor-derived TGF-beta acting on stromal fibroblasts remodels the tumor matrix and induces expression of mitogenic signals towards the carcinoma cells, and upon acting on endothelial cells and pericytes, TGF-beta regulates angiogenesis. Finally, TGF-beta suppresses proliferation and differentiation of lymphocytes including cytolytic T cells, natural killer cells and macrophages, thus preventing immune surveillance of the developing tumor. Current clinical approaches aim at establishing novel cancer drugs whose mechanisms target the TGF-beta pathway. In conclusion, TGF-beta signaling is intimately implicated in tumor development and contributes to all cardinal features of tumor cell biology.
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| 9. |
- Pardali, Katerina, 1974-
(författare)
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Mechanisms of Regulation of the Cell Cycle Inhibitor p21Waf1/Cip1 in TGF-β-Mediated Cell Growth Inhibition
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes.The cell cycle inhibitor p21Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect.The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal p21 promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as Id2 that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene Meox2, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur.This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.
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| 10. |
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| 11. |
- Pardali, Katerina, et al.
(författare)
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Smad pathway-specific transcriptional regulation of the cell cycle inhibitor p21Waf1/Cip1
- 2005
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Ingår i: Journal of Cellular Physiology. - : WILEY-LISS, INC.. - 0021-9541 .- 1097-4652. ; 204:1, s. 260-272
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) inhibits epithelial cell growth, in part via transcriptional induction of the cell cycle inhibitor p21(WAF1/Cip1) (p21). We show that bone morphogenetic protein (BMP)-7 induces higher p21 expression than TGF-beta1 in various epithelial cells. Despite this, BMP-7 only weakly suppresses epithelial cell proliferation, as Id2, a cell cycle-promoting factor, becomes concomitantly induced by BMP-7. Signaling studies with all type I receptors of the TGF-beta superfamily show that BMP receptors induce higher p21 expression than TGF-beta/activin receptors. Smad4 is essential for p21 regulation by all receptor pathways. Based on the previously known ability of c-Myc to block p21 expression and epithelial growth arrest in response to TGF-beta1, we demonstrate that ectopic c-Myc expression can abrogate Smad-mediated p21 induction by all TGF-beta and BMP receptors. Furthermore, p21 induction by all receptor pathways can be blocked by the natural inhibitors of the TGF-beta superfamily. Smad7 inhibits all pathways whereas Smad6 selectively inhibits the BMP pathways. The observed pathway specificity reflects the efficiency by which BMP Smads, compared to TGF-beta Smads, transactivate the p21 promoter. In addition, BMP-specific Smads, Smad1, Smad5, and especially Smad8, induce endogenous p21 mRNA and protein levels, while they fail to induce epithelial growth inhibition when compared to TGF-beta receptor-phosphorylated Smads (R-Smads), Smad2 and Smad3. Thus, p21 is a common target of all TGF-beta superfamily pathways. However, the ability of TGF-beta superfamily members to induce cell growth arrest depends on the regulation of additional gene targets.
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| 12. |
- Pinidiyaarachchi, Amalka, et al.
(författare)
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A detailed analysis of 3D subcellular signal localization
- 2009
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Ingår i: Cytometry Part A. - : Wiley. - 1552-4922 .- 1552-4930. ; 75A:4, s. 319-328
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Tidskriftsartikel (refereegranskat)abstract
- Detection and localization of fluorescent signals in relation to other subcellular structures is an important task in various biological studies. Many methods for analysis of fluorescence microscopy image data are limited to 2D. As cells are in fact 3D structures, there is a growing need for robust methods for analysis of 3D data. This article presents an approach for detecting point-like fluorescent signals and analyzing their subnuclear position. Cell nuclei are delineated using marker-controlled (seeded) 3D watershed segmentation. User-defined object and background seeds are given as input, and gradient information defines merging and splitting criteria. Point-like signals are detected using a modified stable wave detector and localized in relation to the nuclear membrane using distance shells. The method was applied to a set of biological data studying the localization of Smad2-Smad4 protein complexes in relation to the nuclear membrane. Smad complexes appear as early as 1 min after stimulation while the highest signal concentration is observed 45 min after stimulation, followed by a concentration decrease. The robust 3D signal detection and concentration measures obtained using the proposed method agree with previous observations while also revealing new information regarding the complex formation.
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| 13. |
- Valcourt, Ulrich, et al.
(författare)
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Functional role of Meox2 during the epithelial cytostatic response to TGF-beta
- 2007
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Ingår i: Molecular Oncology. - : Wiley. - 1878-0261 .- 1574-7891. ; 1:1, s. 55-71
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor beta (TGF-beta) suppresses epithelial cell growth. We have identified a new target gene of the TGF-beta/Smad pathway, Meox2, encoding the homeodomain transcription factor that is known to regulate endothelial cell proliferation and muscle development. Knockdown of endogenous Meox2 by RNA interference prevented the TGF-beta1-induced cytostatic response. Moreover, ectopic Meox2 suppressed epithelial cell proliferation in cooperation with TGF-beta1, and mediated induction of the cell cycle inhibitor gene p21. Transcriptional induction of p21 by Meox2 required a distal region of the p21 promoter that spans the p53-binding site. We show that Meox2 can form protein complexes with Smads leading to cooperative regulation of p21 gene expression. Finally, we found that in cell models that undergo both cell cycle arrest and epithelial-mesenchymal transition (EMT), ectopic Meox2 failed to induce EMT and inhibited the proper EMT response to TGF-beta. Thus, Meox2 is primarily involved in the TGF-beta tumor suppressor pathway.
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| 14. |
- Winkler, Carla, et al.
(författare)
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Activation of group 2 innate lymphoid cells after allergen challenge in asthmatic patients
- 2019
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Ingår i: Journal of Allergy and Clinical Immunology. - : MOSBY-ELSEVIER. - 0091-6749 .- 1097-6825. ; 144:1, s. 61-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Group 2 innate lymphoid cells (ILC2s) are effective producers of IL-5 and IL-13 during allergic inflammation and bridge the innate and adaptive immune responses. ILC2 numbers are increased in asthmatic patients compared with healthy control subjects. Thus far, human data describing their phenotype during acute allergic inflammation in the lung are incomplete. Objectives: This study aims to characterize and compare blood and lung-derived ILC2s before and after segmental allergen challenge in patients with mild-to-moderate asthma with high blood eosinophil counts (amp;gt;= 300 cells/mu L). Methods: ILC2s were isolated from blood and bronchoalveolar lavage (BAL) fluid before and after segmental allergen challenge. Cells were sorted by means of flow cytometry, cultured and analyzed for cytokine release or migration, and sequenced for RNA expression. Results: ILC2s were nearly absent in the alveolar space under baseline conditions, but numbers increased significantly after allergen challenge (P amp;lt; .05), whereas at the same time, ILC2 numbers in blood were reduced (P amp;lt; .05). Prostaglandin D2 and CXCL12 levels in BAL fluid correlated with decreased ILC2 numbers in blood (P = .004, respective P = .024). After allergen challenge, several genes promoting type 2 inflammation were expressed at greater levels in BAL fluid compared with blood ILC2s, whereas blood ILC2s remain unactivated. Conclusion: ILC2s accumulate at the site of allergic inflammation and are recruited from the blood. Their transcriptional and functional activation pattern promotes type 2 inflammation.
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| 15. |
- Zieba, Agata, et al.
(författare)
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Bright-Field Microscopy Visualization of Proteins and Protein Complexes by In Situ Proximity Ligation with Peroxidase Detection
- 2010
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 56:1, s. 99-110
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research. METHODS: We used horseradish peroxidase (HRP)conjugated oligonucleotides to couple in situ PLA with enzymatic visualization of the localized detection event. RESULTS: We demonstrate the detection of protein complexes, both in cells and in tissue sections, and show that we can quantify the complexes with image-analysis software specially developed for recognizing HRP signals in bright-field microscopy images. We show that fluorescence and HRP signals produce equivalent results, both ill cultured cells and in tissue samples. CONCLUSIONS: The combination of in situ PLA with bright-field detection and automated image analysis allows the signals present to be Counted in an automated fashion and thus provides a sensitive and specific method for quantification of proteins and protein complexes with bright-field microscopy. With this approach, in situ PLA can be used without the requirement for expensive fluorescence microscopes, thereby avoiding problems with nonspecific fluorescence while maintaining compatibility with conventional histologic staining.
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| 16. |
- Zieba, Agata, et al.
(författare)
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Intercellular variation in signaling through the TGF-β pathway and its relation to cell densityand cell cycle phase
- 2012
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Ingår i: Molecular & Cellular Proteomics. - : American Society for Biochemistry and Molecular Biology. - 1535-9476 .- 1535-9484. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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