Decreased apopto-phagocytic gene expression in the macrophages of systemic lupus erythematosus patients.

The clearance of apoptotic cells has an important role in the maintenance of tissue homeostasis and in the protection of tissues from the inflammatory and immunogenic contents of dying cells. A defect in the recognition and phagocytosis of apoptotic cells contributes to the development of chronic inflammation and autoimmune disorders. We have observed that compared with healthy donors, differentiated macrophages from patients with untreated systemic lupus erythematosus (SLE) showed decreased phagocytosis of apoptotic neutrophils. A TaqMan Low Density Array was designed to determine the mRNA expression levels of 95 apopto-phagocytic genes in differentiated non-phagocytosing and phagocytosing macrophages. In the macrophages of clinically and immunoserologically active SLE patients, 39 genes were expressed at lower levels than in the control macrophages. When inactive patients were compared with those with minor immunoserological abnormalities or patients in an immunoserologically active state, a relationship was observed between the altered gene expression profile and the disease state. In the macrophages of patients with engulfing apoptotic cells, an upregulation of genes involved in inflammation, autophagy, and signaling was observed. These results indicate that novel immune-pathological pathways are involved in SLE and suggest targets for potential therapeutic modulation.

Low concentration Phloxine B staining for high chemical contrast, nonlinear microscope mosaic imaging of skin alterations in pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is an autosomal recessive metabolic disorder characterized by ectopic mineralization of soft connective tissue. Histopathology findings include fragmented, mineralized elastic fibers and calcium deposits in the mid-dermis. Nonlinear microscopy (NLM) can be used for visualization of these histopathological alterations of the mid-dermis in PXE-affected skin sections. Upon introducing a normalized 3D color vector representation of emission spectra of three of the main tissue components (collagen, elastin and calcification) we found that due to their broad, overlapping emission spectra, spectral separation of emission from elastin and calcification is practically impossible in fresh-frozen or unstained, deparaffinized PXE sections. However, we found that the application of a low concentration Phloxine B staining after the deparaffinization process creates an imaging contrast for these two tissue components, which enables spectral decomposition of their fluorescence images. The obtained concentration maps for calcium deposits can be well suited for the determination of illness severity by quantitative analysis.

Human beta 2-microglobulin is a substrate of tissue transglutaminase: polymerization in solution and on the cell surface.

Incubation of purified human beta 2-microglobulin (B2-m) with tissue transglutaminase (Tgase) resulted in the formation of high molecular weight polymers revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of 30 mM [14C]methylamine, the polymer formation was prevented, but incorporation of methylamine into beta 2-m (equal to 1 methylamine per 1 molecule) could be observed. From the sheddings of peripheral blood mononuclear cells occurring in the presence of Tgase, it is apparent that anti-beta 2-m immunoadsorbent removed, in addition to human leukocyte antigen (HLA) and beta 2-m, some other proteins. The enzyme could incorporate [14C]methylamine into beta 2-m of the shedding cells. On addition of rabbit anti-human beta 2-m antibody, followed by fluoresceine-labeled goat anti-rabbit IgG antibody to human mononuclear blood cells, the otherwise homogeneous distribution of fluorescence turned into spots and patches on cells previously incubated with Tgase or Ca2+-ionophore A23187.

Expression of tissue transglutaminase in Balb-C 3T3 fibroblasts: effects on cellular morphology and adhesion.

Tissue transglutaminase is a cytosolic enzyme whose primary function is to catalyze the covalent cross-linking of proteins. To investigate the functions of this enzyme in physiological systems, we have established lines of Balb-C 3T3 fibroblasts stably transfected with a constitutive tissue transglutaminase expression plasmid. Several cell lines expressing high levels of catalytically active tissue transglutaminase have been isolated and characterized. Transglutaminase-transfected cells showed morphologic features quite distinct from their nontransfected counterparts. Many of the cells showed an extended and very flattened morphology that reflected increased adhesion of the cells to the substratum. Other cells, particularly those showing the highest levels of intracellular transglutaminase expression, showed extensive membrane blebbing and cellular fragmentation. The results of these experiments suggest that the induction and activation of tissue transglutaminase may contribute both to changes in cellular morphology and adhesiveness.

Some lessons from the tissue transglutaminase knockout mouse.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-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.

AluI polymorphism of the bovine growth hormone (GH) gene, resumption of ovarian cyclicity, milk production and loss of body condition at the onset of lactation in dairy cows.

Relationships among GH genotype (AluI polymorphism), parity, metritis and interval from calving to first ovulation, milk production and body condition score (BCS) loss were determined in dairy cows (n=307) on four large-scale farms in Hungary. Cows with systemic signs of puerperal metritis or mastitis were excluded. Time of the first postpartum (PP) ovulation was obtained from milk progesterone profiles. Based on GH genotype determination, groups of leucine homozygous cows (n=246) and valine allele carriers (n=61) were formed. All animals became cyclic during the study period. The average interval to first ovulation was 27.6+/-0.69-d PP (mean+/-S.D.). Genotype had no effect on the commencement of ovarian cyclicity. First ovulation occurred sooner after calving in pluriparous than in primiparous cows. The greater BCS loss cows had during the first 30-d PP, the longer they took to resume cyclic ovarian function. The interval from calving to first ovulation was substantially affected by farm, but not by mild cases of puerperal metritis. Genotype was not related to cumulative 30-d milk yield or BCS loss after calving. Primiparous cows had lower milk yield than pluriparous ones. Cows with metritis lost more body condition than healthy individuals in the first month postpartum. We concluded that, under field conditions, AluI polymorphism of the bovine GH gene had no effect on the interval from calving to first ovulation and could not be directly related to differences in milk yield and to the extent of BCS loss during the first month after calving in Holstein-Friesian cows.

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes.

MCF-7 cells undergo autophagic death upon tamoxifen treatment. Plated on non-adhesive substratum these cells died by anoikis while inducing autophagy as revealed by monodansylcadaverine staining, elevated light-chain-3 expression and electron microscopy. Both de novo and anoikis-derived autophagic dying cells were engulfed by human macrophages and MCF-7 cells. Inhibition of autophagy by 3-methyladenine abolished engulfment of cells dying through de novo autophagy, but not those dying through anoikis. Blocking exposure of phosphatidylserine (PS) on both dying cell types inhibited phagocytosis by MCF-7 but not by macrophages. Gene expression profiling showed that though both types of phagocytes expressed full repertoire of the PS recognition and signaling pathway, macrophages could evolve during engulfment of de novo autophagic cells the potential of calreticulin-mediated processes as well. Our data suggest that cells dying through autophagy and those committing anoikis with autophagy may engage in overlapping but distinct sets of clearance mechanisms in professional and non-professional phagocytes.

Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

Tissue transglutaminase-dependent posttranslational modification of the retinoblastoma gene product in promonocytic cells undergoing apoptosis.

The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program.

Effect of MYOG genotypes on growth rate and production traits in Hungarian large white pigs.

The effect of the porcine myogenin (Myog) 3' polymorphism on birth weight, growth rate, carcass weight, lean weight, lean meat percentage and back-fat thickness has been investigated in Hungarian Large White pigs. MYOG genotypes were determined by PCR-RFLP assay. The obtained MYOGA frequency value was 0.6275. Due to the small number of BB piglets the effect of the MYOG genotypes on birth weight was not significant; however, an increasing tendency was observed from genotype AA to BB. The growth rate difference between MYOG genotypes was significant: BB animals showed the highest growth rate values during the fattening period. Since few results are available on the possible use of MYOG gene polymorphism in selection to improve carcass and growth traits, by this study the authors hope to provide additional data on this particular subject.

Clozapine modifies the differentiation program of human adipocytes inducing browning.

Administration of second-generation antipsychotic drugs (SGAs) often leads to weight gain and consequent cardio-metabolic side effects. We observed that clozapine but not six other antipsychotic drugs reprogrammed the gene expression pattern of differentiating human adipocytes ex vivo, leading to an elevated expression of the browning marker gene UCP1, more and smaller lipid droplets and more mitochondrial DNA than in the untreated white adipocytes. Laser scanning cytometry showed that up to 40% of the differentiating single primary and Simpson-Golabi-Behmel syndrome (SGBS) adipocytes had the characteristic morphological features of browning cells. Furthermore, clozapine significantly upregulated ELOVL3, CIDEA, CYC1, PGC1A and TBX1 genes but not ZIC1 suggesting induction of the beige-like and not the classical brown phenotype. When we tested whether browning induced by clozapine can be explained by its known pharmacological effect of antagonizing serotonin (5HT) receptors, it was found that browning cells expressed 5HT receptors 2A, 1D, 7 and the upregulation of browning markers was diminished in the presence of exogenous 5HT. Undifferentiated progenitors or completely differentiated beige or white adipocytes did not respond to clozapine administration. The clozapine-induced beige cells displayed increased basal and oligomycin-inhibited (proton leak) oxygen consumption, but these cells showed a lower response to cAMP stimulus as compared with control beige adipocytes indicating that they are less capable to respond to natural thermogenic anti-obesity cues. Our data altogether suggest that novel pharmacological stimulation of these masked beige adipocytes can be a future therapeutic target for the treatment of SGA-induced weight gain.

Clearance of autophagy-associated dying retinal pigment epithelial cells - a possible source for inflammation in age-related macular degeneration.

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.

Lipids associated with tissue transglutaminase.

A substantial amount of lipids (cholesterol and its esters, mono-, di- and triacylglycerols, free fatty acids and the phospholipids phosphatidylethanolamine and phosphatidylinositol) was found associated with tissue transglutaminase purified to apparent homogeneity from guinea pig liver. Removal of lipids results in an increased tendency of the enzyme for self-association and a decreased stability. Lauric acid was detected following hydroxylamine treatment of the enzyme, suggesting the occurrence of a fatty acid-type, covalent, posttranslational modification of transglutaminase. The results provide support for the idea that part of tissue transglutaminase may be localized in the cell membrane.

The comparative ability of plasma and tissue transglutaminases to use collagen as a substrate.

Heat denatured type I and type III calf skin collagen were found to be substrates for guinea pig liver transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13) but not for active plasma factor XIII (factor XIIIa). Liver transglutaminase was shown to catalyse incorporation of 14C-putrescine into subunits of denatured collagen of both types, cross-linking of the latter into high molecular weight polymers and their co-cross-linking to fibrin and fibrinogen. Factor XIIIa is inactive in these respects. None of these reactions was catalysed by liver transglutaminase and plasma factor XIIIa when nondenatured collagens both soluble or in the forms of reconstituted fibrils served as substrates. Some cross-linking of cleavage products of collagen type I (obtained by treatment with collagenase from human neutrophiles) was induced by liver transglutaminase and factor XIIIa. The results indicate that although appropriate glutamine and lysine residues for a epsilon-(gamma-glutamine) lysine cross-linked formation are present in collagen, the native conformation of collagen prevents the action of liver transglutaminase and factor XIIIa.

Cleavage of thrombosthenin A by thrombin. Evidence for the existence of two types of bovine platelet actin.

Bovin platelet actin prepared by Spudich's method (Spudich, J. A. (1972) Cold Spring Harbor Symp. Quant. Biol. 27, 585-594) separated into two peaks on a Sephadex G-200 column. The actin of both peaks had a mol. wt. of 42 000 on sodium dodecyl sulfate-polyacrylamide gel and activated myosin ATPase, although in a quantitatively different manner. Actin eluted in the first peak (probably an oligomeric form) was not polymerized in 2 mM MgCl2 and 0.05 M KCl, while that of the second peak went through normal G-F transformation. If CaATP was present in the incubation mixture neither actin was attacked by thrombin. However, if EDTA was added, thrombin split G-actins and the pattern of cleavage was the same as that found for muscle actin in our earlier studies, i.e. the final split products were two actinopeptides and two larger fragments of 26 500 and 11 000 daltons. It is suggested that the possible attraction of membrane-associated platelet actin for thrombin may have an importance in thrombin-induced platelet aggregation.

The presence of a covalently cross-linked matrix in human platelets.

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.

Evaluation of phenylthiourea derivatives as inhibitors of transglutaminase-catalyzed reaction in Chinese hamster ovary cells.

1-(5-Aminopentyl)-3-phenylthiourea (PPTU), a recently developed inhibitor of the transglutaminase-catalyzed reaction (K.N. Lee, L. Fesus, S.T. Yancey, J.E. Girard, and S.I. Chung, (1985) J. Biol. Chem. 260, 14689-14694) was evaluated as a possible probe to examine the physiological role of transglutaminase (EC 2.3.2.13) in Chinese hamster ovary (CHO) cells. The [14C]PPTU in cell culture was readily taken up by CHO cells and was found to be covalently attached to high-molecular-weight proteins which are associated with the particulate fractions. Incubating cell homogenates, in the presence of Ca2+, incorporated the labeled PPTU exclusively into high-molecular-weight proteins that were also undergoing polymerization. PPTU at 0.1 mM, a concentration close to the Ki value reported for inhibition of tissue transglutaminase-catalyzed amine incorporation into the B chain of oxidized insulin, decreased high-molecular-weight protein polymerization, whereas PPTU at the same concentrations showed no effect on CHO cell proliferation or on in vitro calmodulin activation of cyclic nucleotide phosphodiesterase. These results suggest that transglutaminase may not be a constitutive enzyme in cell proliferation.

In vivo and in vitro induction of 'tissue' transglutaminase in rat hepatocytes by retinoic acid.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.

Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors.

Vitamin A deficiency has been known for a long time to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Increasing evidence suggests that retinoic acids derived from vitamin A are involved in the functional regulation of the immune system. Of the two groups of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) all-trans and 9-cis retinoic acids are high affinity ligands for RARs and 9-cis retinoic acid additionally binds to RXRs. In cells, at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid by unknown mechanisms. Apoptosis plays a major role in shaping the T cell repertoire and one way in which retinoids may affect immune functions is to influence the various apoptosis pathways. Indeed, it has been shown that retinoic acids can induce apoptosis, increase the rate of dexamethasone-induced death and inhibit activation-induced death of thymocytes and T lymphocytes. Therefore, retinoids together with glucocorticoids may be involved in regulating positive and negative selection of T lymphocytes. Here we demonstrate that retinoids can induce apoptosis of T cells through the stimulation of RARgamma. Specific stimulation of RARalpha, on the other hand, prevents both RARgamma-dependent and TCR-mediated cell death. In all these functions 9-cis retinoic acid proved to be more effective than all-trans retinoic acid suggesting the involvement of RXRs. Based on these results a possible mechanism through which costimulation of RARs and RXRs might affect spontaneous and activation-induced death of T lymphocytes is proposed.

Apoptosis-linked in vivo regulation of the tissue transglutaminase gene promoter.

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.