Zoltán Balajthy

1,25-Dihydroxyvitamin D3 Is an autonomous regulator of the transcriptional changes leading to a tolerogenic dendritic cell phenotype

Activation of vitamin D receptor (VDR) by 1,25-dihydroxyvitamin D3 (1,25-vitD) reprograms dendritic cells (DC) to become tolerogenic. Previous studies suggested that 1,25-vitD could inhibit the changes brought about by differentiation and maturation of DCs. Underpinning the described phenotypic and functional alterations, there must be 1,25-vitD-coordinated transcriptional events. However, this transcriptional program has not been systematically investigated, particularly not in a developmental context. Hence, it has not been explored how 1,25-vitD-regulated genes, particularly the ones bringing about the tolerogenic phenotype, are connected to differentiation. We conducted global gene expression analysis followed by comprehensive quantitative PCR validation to clarify the interrelationship between 1,25-vitD and differentiation-driven gene expression patterns in developing human monocyte-derived and blood myeloid DCs. In this study we show that 1,25-vitD regulates a large set of genes that are not affected by differentiation. Interestingly, several genes, impacted both by the ligand and by differentiation, appear to be regulated by 1,25-vitD independently of the developmental context. We have also characterized the kinetics of generation of 1,25-vitD by using three early and robustly regulated genes, the chemokine CCL22, the inhibitory receptors CD300LF and CYP24A1. We found that monocyte-derived DCs are able to turn on 1,25-vitD sensitive genes in early phases of differentiation if the precursor is present. Our data collectively suggest that exogenous or endogenously generated 1,25-vitD regulates a large set of its targets autonomously and not via inhibition of differentiation and maturation, leading to the previously characterized tolerogenic state.

Transglutaminase induction by various cell death and apoptosis pathways.

Clarification of the molecular details of forms of natural cell death, including apoptosis, has become one of the most challenging issues of contemporary biomedical sciences. One of the effector elements of various cell death pathways is the covalent cross-linking of cellular proteins by transglutaminases. This review will discuss the accumulating data related to the induction and regulation of these enzymes, particularly of tissue type transglutaminase, in the molecular program of cell death. A wide range of signalling pathways can lead to the parallel induction of apoptosis and transglutaminase, providing a handle for better understanding the exact molecular interactions responsible for the mechanism of regulated cell death.

Transglutaminase 2 Is Needed for the Formation of an Efficient Phagocyte Portal in Macrophages Engulfing Apoptotic Cells

Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin β3. We have previously shown that TG2−/− mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin β3, a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin β3 to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin β3 and Rac1. In the absence of TG2, integrin β3 cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross‐linking enzyme, which forms isopeptide bonds between protein‐linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine‐donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine‐donor substrate. Twenty‐six Gln‐containing sequences from the second and third biopanning rounds were susceptible for TG2‐mediated incorporation of 5‐(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage‐selected sequences, and the N‐terminal glutamine‐rich domain of SWI1/SNF1‐related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry‐based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q6, Q8, and Q22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (KMapp = 250 μM, kcat = 18.3 sec−1, and kcat/KMapp = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full‐length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro.

Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin and adiponectin, suggesting that both glucose and fat metabolism may be affected by these drugs. These data further suggest that antipsychotic treatments in patients alter the gene expression patterns in adipocytes in a coordinated fashion and priming them for a low-level inflammatory state.

Some lessons from the tissue transglutaminase knockout mouse.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications. It acts as a G protein in transmembrane signaling and as a cell surface adhesion mediator, this distinguishes it from other members of the transglutaminase family. The sequence motifs and domains revealed in the TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion, and cell death. Though many biological functions of the enzyme have already been described or proposed previously, studies of TG2 null mice by our laboratory during the past years revealed several novel in vivo roles of the protein. In this review we will discuss these novel roles in their biological context.

Tissue transglutaminase contributes to the all-trans-retinoic acid-induced differentiation syndrome phenotype in the NB4 model of acute promyelocytic leukemia.

Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation-related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNA interference-mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions, and their silencing lead to reduced adhesive, migratory, and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell-cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, CCL3, CCL22, CCL24, and cytokines IL1B and IL8 involved in the development of differentiation syndrome are expressed at significantly lower level in TG2-KD NB4 than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of differentiation syndrome.

High content analysis of differentiation and cell death in human adipocytes

Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high‐content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide‐based image cytometry and image processing algorithms were used and optimized for high‐throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9–14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFα)‐induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program.

Interaction of differentiated human adipocytes with macrophages leads to trogocytosis and selective IL-6 secretion

Obesity leads to adipose tissue inflammation that is characterized by increased release of proinflammatory molecules and the recruitment of activated immune cells. Although macrophages are present in the highest number among the immune cells in obese adipose tissue, not much is known about their direct interaction with adipocytes. We have introduced an ex vivo experimental system to characterize the cellular interactions and the profile of secreted cytokines in cocultures of macrophages and human adipocytes differentiated from either mesenchymal stem cells or a preadipocyte cell line. As observed by time-lapse microscopy, flow, and laser-scanning cytometry, macrophages phagocytosed bites of adipocytes (trogocytosis), which led to their de novo, phagocytosis and NF-κB-dependent synthesis, then release of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1. IL-6 secretion was not accompanied by secretion of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and IL-8, except MCP-1. LPS-induced release of TNF-α, IL-8 and MCP-1 was decreased in the presence of the differentiated adipocytes but the IL-6 level did not subside suggesting that phagocytosis-dependent IL-6 secretion may have significant regulatory function in the inflamed adipose tissue.

Protein cross-linking by chlorinated polyamines and transglutamylation stabilizes neutrophil extracellular traps

Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.