Phenotype-specific therapeutic efficacy of ilofotase alfa in patients with sepsis-associated acute kidney injury.

BACKGROUND: There is no effective treatment for sepsis-associated acute kidney injury (SA-AKI). Ilofotase alfa (human recombinant alkaline phosphatase) has been shown to exert reno-protective properties, although it remains unclear which patients might be most likely to benefit. We aimed to identify a clinical phenotype associated with ilofotase alfa's therapeutic efficacy. METHODS: Data from 570 out of 650 patients enrolled in the REVIVAL trial were used in a stepwise machine learning approach. First, clinical variables with increasing or decreasing risk ratios for ilofotase alfa treatment across quartiles for the main secondary endpoint, Major Adverse Kidney Events up to day 90 (MAKE90), were selected. Second, linear regression analysis was used to determine the therapeutic effect size. Finally, the top-15 variables were used in different clustering analyses with consensus assessment. RESULTS: The optimal clustering model comprised two phenotypes. Phenotype 1 displayed relatively lower disease severity scores, and less pronounced renal and pulmonary dysfunction. Phenotype 2 exhibited higher severity scores and creatinine, with lower eGFR and bicarbonate levels. Compared with placebo treatment, ilofotase alfa significantly reduced MAKE90 events for phenotype 2 patients (54% vs. 68%, p = 0.013), but not for phenotype 1 patients (49% vs. 46%, p = 0.54). CONCLUSION: We identified a clinical phenotype comprising severely ill patients with underlying kidney disease who benefitted most from ilofotase alfa treatment. This yields insight into the therapeutic potential of this novel treatment in more homogeneous patient groups and could guide patient selection in future trials, showing promise for personalized medicine in SA-AKI and other complex conditions.

A Phase 2a, Double-Blind, Placebo-controlled Randomized Trial of Inhaled TLR9 Agonist AZD1419 in Asthma.

Rationale: To examine the potential of TLR9 (Toll-like receptor 9) activation to modulate the type 2 immune response in asthma.Objectives: To evaluate efficacy and safety of AZD1419, an inhaled TLR9 agonist, in a phase 2a, randomized, double-blind trial.Methods: Adult patients with asthma with a history of elevated eosinophils (>250 cells/µl) were randomized 1:1 to receive 13 once-weekly doses of inhaled AZD1419 (1, 4, or 8 mg; n = 40) or placebo (n = 41). Inhaled corticosteroids and long-acting ß2-agonist were tapered down and then discontinued. The last four doses of AZD1419 were given without maintenance medication, followed by a 40-week observation period. Primary endpoint was time to loss of asthma control (LOC).Measurements and Main Results: AZD1419 induced a T-helper cell type 1-type IFN response with a sustained reduction in markers of type 2 inflammation. However, there were no statistically significant differences between AZD1419 and placebo for time to LOC, proportion of patients with LOC, changes in Asthma Control Questionnaire-five-item version, exacerbations, reliever use, FEV1, peak expiratory flow, or fractional exhaled nitric oxide (FeNO). LOC was predicted by an early rise in FeNO in 63% of patients. Despite withdrawal of maintenance treatment, 24 patients completed the study without LOC; AZD1419 n = 11, placebo n = 13. Adverse events were balanced across groups, with no deaths or serious adverse events judged as causally related to AZD1419.Conclusions: AZD1419 was safe and well tolerated but did not lead to improved asthma control, despite reducing markers of type 2 inflammation. Results suggest that a novel accelerated step-down approach based on FeNO is possible for patients with well-controlled asthma.

Expression of c-Kit discriminates between two functionally distinct subsets of human type 2 innate lymphoid cells.

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-Kithi and c-Kitlo ). When examined for their potential for functional plasticity we found that c-Kitlo ILC2 displayed greater potential to produce type 2 cytokines, possibly representing fully mature and lineage committed ILC2. On the other hand, c-Kithi 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-Kithi ILC2 produced higher levels of IFN-γ than c-Kitlo 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.

Activation of group 2 innate lymphoid cells after allergen challenge in asthmatic patients.

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 (≥300 cells/µL). 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 < .05), whereas at the same time, ILC2 numbers in blood were reduced (P < .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.

IL-33 regulates cytokine production and neutrophil recruitment via the p38 MAPK-activated kinases MK2/3.

IL-33 is an IL-1-related cytokine that can act as an alarmin when released from necrotic cells. Once released, it can target various immune cells including mast cells, innate lymphoid cells and T cells to elicit a Th2-like immune response. We show here that bone marrow-derived mast cells produce IL-13, IL-6, TNF, GM-CSF, CCL3 and CCL4 in response to IL-33 stimulation. Inhibition of the p38 MAPK, or inhibition or knockout of its downstream kinases MK2 and MK3, blocked the production of these cytokines in response to IL-33. The mechanism downstream of MK2/3 was cytokine specific; however, MK2 and MK3 were able to regulate TNF and GM-CSF mRNA stability. Previous studies in macrophages have shown that MK2 regulates mRNA stability via phosphorylation of the RNA-binding protein TTP (Zfp36). The regulation of cytokine production in mast cells was, however, independent of TTP. MK2/3 were able to phosphorylate the TTP-related protein Brf1 (Zfp36 l1) in IL-33-stimulated mast cells, suggesting a mechanism by which MK2/3 might control mRNA stability in these cells. In line with its ability to regulate in vitro IL-33-stimulated cytokine production, double knockout of MK2 and 3 in mice prevented neutrophil recruitment following intraperitoneal injection of IL-33.

Polymorphisms in the multidrug-resistance 1 gene related to glucocorticoid response in rheumatoid arthritis treatment.

A substantial proportion of rheumatoid arthritis (RA)-patients experience an insufficient response to glucocorticoids, an important therapeutic agent in RA. The multidrug-resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is an efflux pump that actively transports substrates, such as glucocorticoids, out of the cell. We investigated if the variation in response might be explained by single-nucleotide polymorphisms (SNPs) in the MDR1 gene. RA-patients treated with intravenous methylprednisolone pulses (n = 18) or oral prednisone/prednisolone (n = 22) were included in a prospective cohort, and clinical response was measured after 5 and 30 days, respectively. The C1236T, G2677A/T, and C3435T SNPs were determined, and the functionality of P-gp was assessed by flow cytometry (Rhodamine efflux assay). Carriage of the G2677A/T SNP was significantly associated with response (OR = 6.18, p = 0.035), the other SNPs showed trends. Stratified for received treatment, the effect was only present in methylprednisolone treated patients. Mutant allele carriage significantly decreased functionality of P-gp in B cells, though had a smaller impact in other PBMC subtypes. Carriage of a MDR1 SNP was related to a response to methylprednisolone in this study, which his suggests that RA-patients carrying wild-type alleles might benefit from P-gp inhibition or administration of glucocorticoid analogues that are non-P-gp substrates.

Intercellular variation in signaling through the TGF-ß 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 ß-induced Smad interactions, providing essential information about TGF-ß 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-ß 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.

Notch signaling is necessary for epithelial growth arrest by TGF-beta.

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.

Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection.

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 in 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.

p38 MAPK signalling regulates cytokine production in IL-33 stimulated Type 2 Innate Lymphoid cells.

Type 2 Innate lymphoid cells (ILC2s) are implicated in helminth infections and asthma where they play a role in the production of Th2-type cytokines. ILC2s express the IL-33 receptor and are a major cell type thought to mediate the effects of this cytokine in vivo. To study the signalling pathways that mediate IL-33 induced cytokine production, a culture system was set up to obtain pure populations of ILC2s from mice. Inhibitors of the p38α/ß and ERK1/2 MAPK pathways reduced the production of IL-5, IL-6, IL-9, IL-13 and GM-CSF by ILC2 in response to IL-33, with inhibition of p38 having the greatest effect. MK2 and 3 are kinases activated by p38α; MK2/3 inhibitors or knockout of MK2/3 in mice reduced the production of IL-6 and IL-13 (two cytokines implicated in asthma) but not IL-5, IL-9 or GM-CSF in response to IL-33. MK2/3 inhibition also suppressed IL-6 and IL-13 production by human ILC2s. MK2/3 were required for maximal S6 phosphorylation, suggesting an input from the p38α-MK2/3 pathway to mTOR1 activation in ILC2s. The mTORC1 inhibitor rapamycin also reduced IL-6 and IL-13 production, which would be consistent with a model in which MK2/3 regulate IL-6 and IL-13 via mTORC1 activation in ILC2s.

A MALT1 inhibitor suppresses human myeloid DC, effector T-cell and B-cell responses and retains Th1/regulatory T-cell homeostasis.

The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or T-cell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-É£) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-É£, in a mouse model of acute T-cell activation, and long-term treatment did not lead to an increase in IFN-É£ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases.

Immunometabolism pathways as the basis for innovative anti-viral strategies (INITIATE): A Marie Sklodowska-Curie innovative training network.

The past century has witnessed major advances in the control of many infectious diseases, yet outbreaks and epidemics caused by (re-) emerging RNA viruses continue to pose a global threat to human health. As illustrated by the global COVID19 pandemic, high healthcare costs, economic disruption and loss of productivity reinforce the unmet medical need to develop new antiviral strategies to combat not only the current pandemic but also future viral outbreaks. Pivotal for effective anti-viral defense is the innate immune system, a first line host response that senses and responds to virus infection. While molecular details of the innate immune response are well characterized, this research field is now being revolutionized with the recognition that cell metabolism has a major impact on the antiviral and inflammatory responses to virus infections. A detailed understanding of the role of metabolic regulation with respect to antiviral and inflammatory responses, together with knowledge of the strategies used by viruses to exploit immunometabolic pathways, will ultimately change our understanding and treatment of pathogenic viral diseases. INITIATE is a Marie Sklodowska-Curie Actions Innovative Training Network (MSCA-ITN), with the goal to train 15 early stage PhD researchers (ESRs) to become experts in antiviral immunometabolism (https://initiate-itn.eu/). To this end, INITIATE brings together a highly complementary international team of academic and corporate leaders from 7 European countries, with outstanding track records in the historically distinct research fields of virology, immunology and metabolism. The ESRs of INITIATE are trained in these interdisciplinary research fields through individual investigator-driven research projects, specialized scientific training events, workshops on academia-industry interactions, outreach & communication. INITIATE will deliver a new generation of creative and entrepreneurial researchers who will be able to face the inevitable future challenges in combating viral diseases.

A detailed analysis of 3D subcellular signal localization.

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.

An Angle on MK2 Inhibition-Optimization and Evaluation of Prevention of Activation Inhibitors.

The mitogen-activated protein kinase p38α pathway has been an attractive target for the treatment of inflammatory conditions such as rheumatoid arthritis. While a number of p38α inhibitors have been taken to the clinic, they have been limited by their efficacy and toxicological profile. A lead identification program was initiated to selectively target prevention of activation (PoA) of mitogen-activated protein kinase-activated protein kinase 2 (MK2) rather than mitogen- and stress-activated protein kinase 1 (MSK1), both immediate downstream substrates of p38α, to improve the efficacy/safety profile over direct p38α inhibition. Starting with a series of pyrazole amide PoA MK2 inhibitor leads, and guided by structural chemistry and rational design, a highly selective imidazole 9 (2-(3'-(2-amino-2-oxoethyl)-[1,1'-biphenyl]-3-yl)-N-(5-(N,N-dimethylsulfamoyl)-2-methylphenyl)-1-propyl-1H-imidazole-5-carboxamide) and the orally bioavailable imidazole 18 (3-methyl-N-(2-methyl-5-sulfamoylphenyl)-2-(o-tolyl)imidazole-4-carboxamide) were discovered. The PoA concept was further evaluated by protein immunoblotting, which showed that the optimized PoA MK2 compounds, despite their biochemical selectivity against MSK1 phosphorylation, behaved similarly to p38 inhibitors in cellular signaling. This study highlights the importance of selective tool compounds in untangling complex signaling pathways, and although 9 and 18 were not differentiated from p38α inhibitors in a cellular context, they are still useful tools for further research directed to understand the role of MK2 in the p38α signaling pathway.

Actions of TGF-beta as tumor suppressor and pro-metastatic factor in human cancer.

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.

The development of AZD7624 for prevention of exacerbations in COPD: a randomized controlled trial.

Background: p38 mitogen-activated protein kinase (MAPK) plays a central role in the regulation and activation of pro-inflammatory mediators. COPD patients have increased levels of activated p38 MAPK, which correlate with increased lung function impairment, alveolar wall inflammation, and COPD exacerbations. Objectives: These studies aimed to assess the effect of p38 inhibition with AZD7624 in healthy volunteers and patients with COPD. The principal hypothesis was that decreasing lung inflammation via inhibition of p38α would reduce exacerbations and improve quality of life for COPD patients at high risk for acute exacerbations. Methods: The p38 isoform most relevant to lung inflammation was assessed using an in situ proximity ligation assay in severe COPD patients and donor controls. Volunteers aged 18-55 years were randomized into the lipopolysaccharide (LPS) challenge study, which investigated the effect of a single dose of AZD7624 vs placebo on inflammatory biomarkers. The Proof of Principle study randomized patients aged 40-85 years with a diagnosis of COPD for >1 year to AZD7624 or placebo to assess the effect of p38 inhibition in decreasing the rate of exacerbations. Results: The p38 isoform most relevant to lung inflammation was p38α, and AZD7624 specifically inhibited p38α and p38ß isoforms in human alveolar macrophages. Thirty volunteers were randomized in the LPS challenge study. AZD7624 reduced the increase from baseline in sputum neutrophils and TNF-α by 56.6% and 85.4%, respectively (p<0.001). In the 213 patients randomized into the Proof of Principle study, there was no statistically significant difference between AZD7624 and placebo when comparing the number of days to the first moderate or severe exacerbation or early dropout. Conclusion: Although p38α is upregulated in the lungs of COPD patients, AZD7624, an isoform-specific inhaled p38 MAPK inhibitor, failed to show any benefit in patients with COPD.

Nuclear factor YY1 inhibits transforming growth factor beta- and bone morphogenetic protein-induced cell differentiation.

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.

Mechanisms of TGF-beta signaling in regulation of cell growth and differentiation.

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.

Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

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.

Functional role of Meox2 during the epithelial cytostatic response to TGF-beta.

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.