Thymus hormones as prospective anti-inflammatory agents.

IMPORTANCE OF THE FIELD: Inflammatory diseases are characterized by severe immune imbalances, leading to excessive or inappropriate release of mediators, which, in turn, result in massive damage to organs and systems. Effective means to control inappropriate immune reactions are often life-critical needs. Available data on the role of thymus-derived hormones in inflammation show their great potential. AREAS COVERED IN THIS REVIEW: The review aims to systematize information for the last two decades on immune system regulation by thymic peptide hormones, with a primary focus on the role of these hormones in the systemic inflammatory response and inflammatory diseases. Anti-inflammatory potential of three thymic hormones - thymulin, thymosin-alpha, and thymopoietin - is discussed, reviewing recently published clinical and experimental studies. WHAT THE READER WILL GAIN: Our analysis revealed the regulation of inflammatory processes via thymic hormones that could be prospective for therapeutic application. This regulation may be mediated through thymic hormone effects on peripheral immune cell activities and bidirectional coupling between thymic hormones and the hypothalamic-pituitary-adrenal axis. TAKE-HOME MESSAGE: In view of the role of thymic hormones in immune and neuroendocrine systems, they could be suitable as therapeutic agents for inflammation.

The inner nuclear membrane protein emerin regulates beta-catenin activity by restricting its accumulation in the nucleus.

Emerin is a type II inner nuclear membrane (INM) protein of unknown function. Emerin function is likely to be important because, when it is mutated, emerin promotes both skeletal muscle and heart defects. Here we show that one function of Emerin is to regulate the flux of beta-catenin, an important transcription coactivator, into the nucleus. Emerin interacts with beta-catenin through a conserved adenomatous polyposis coli (APC)-like domain. When GFP-emerin is expressed in HEK293 cells, beta-catenin is restricted to the cytoplasm and beta-catenin activity is inhibited. In contrast, expression of an emerin mutant, lacking its APC-like domain (GFP-emerinDelta), dominantly stimulates beta-catenin activity and increases nuclear accumulation of beta-catenin. Human fibroblasts that are null for emerin have an autostimulatory growth phenotype. This unusual growth phenotype arises through enhanced nuclear accumulation and activity of beta-catenin and can be replicated in wild-type fibroblasts by transfection with constitutively active beta-catenin. Our results support recent findings that suggest that INM proteins can influence signalling pathways by restricting access of transcription coactivators to the nucleus.

Dependence of diffusional mobility of integral inner nuclear membrane proteins on A-type lamins.

Integral proteins of the nuclear envelope inner membrane have been proposed to reach their sites by diffusion after their co-translational insertion in the rough endoplasmic reticulum. They are then retained in the inner nuclear membrane by binding to nuclear structures. One such structure is the nuclear lamina, an intermediate filament meshwork composed of A-type and B-type lamin proteins. Emerin, MAN1, and LBR are three integral inner nuclear membrane proteins. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts from wild-type mice and Lmna -/- mice, which lack A-type lamins. We then studied the diffusional mobilities of emerin, MAN1, and LBR using fluorescence recovery after photobleaching. We show that emerin and MAN1, but not LBR, are more mobile in the inner nuclear membrane of cells from Lmna -/- mice than in cells from wild-type mice. In cells from Lmna -/- mice expressing exogenous lamin A, the protein mobilities were similar to those in cells from wild-type mice. This supports a model where emerin and MAN1 are at least partly retained in the inner nuclear membrane by binding to A-type lamins, while LBR depends on other binding partners for its retention.

HIV protease inhibitors block adipocyte differentiation independently of lamin A/C.

OBJECTIVES: To determine the importance of lamin A/C for fat cell differentiation in vitro and for the anti-adipogenic activity of HIV protease inhibitors such as indinavir. METHODS: Lipodystrophy-associated and processing-defective mutants of lamin A were stably expressed at high levels in 3T3-L1 pre-adipocytes. Additionally, 3T3-L1 pre-adipocytes with stable reduction of lamin A/C or emerin were derived. The cells were differentiated for 8 days into mature adipocytes in the presence or absence of indinavir or nelfinavir. RESULTS: 3T3-L1 cells stably expressing high levels of lipodystrophy-associated or processing-defective mutants of lamin A differentiated with comparable efficiencies to control cells. Similarly, cells with dramatically reduced lamin A levels differentiated as efficiently as controls. Although indinavir stimulated the accumulation of unprocessed lamin A, cells with dramatically reduced lamin A/C levels and no detectable prelamin A remained responsive to an indinavir-induced inhibition of adipogenesis. CONCLUSIONS: The ability of HIV protease inhibitor to stimulate the accumulation of unprocessed lamin A is neither necessary nor sufficient to explain their anti-adipogenic activity. Furthermore, lamin A/C plays a minimal role in the differentiation of 3T3-L1.

Changes in thymopoiesis in myasthenia gravis.

This study was undertaken to investigate T-cell maturation in hyperplastic thymi of patients suffering from myasthenia gravis (MG). For this purpose, the expression of the major differentiational molecules (CD4, CD8, and CD3/TCRalphabeta) and that of the regulatory and activation molecules on thymocytes from MG patients and control subjects were estimated by flow cytometric analysis. In the MG patients the increase in relative proportion of immature (CD4-8- TCRalphabeta-) and the most mature (CD4+8- TCRalphabetahigh and CD4-8- TCRhigh encompassing immunoregulatory NKT) thymocytes followed by a decrease in that of CD4+8+CD3-/TCRalphabeta- cells was found. Furthermore, in these patients the relative proportion of CD4+HLA-DR+ and CD4+71+ cells was increased, whereas that of CD4+25+ cells was slightly, but significantly, decreased (reflecting, most likely, decreased contribution of T reg cells bearing this phenotype). Moreover, in MG thymi the percentage of CD45RA+ cells was reduced indicating changes in the selection processes. In keeping with this finding the reduced thymocyte apoptotic index and percentage of cells bearing apoptosing (CD4-8- TCRalphabetalow) phenotype were detected. In conclusion, the study demonstrates substantial changes in intrathymic differentiation of T cells in hyperplastic MG thymi and suggests alterations in selection events providing an increased escape of potentially autoreactive T-cell clones, on one side, and an altered maturation and/or selection of immunoregulatory cells (NKT and CD4+8-25+ T reg cells) keeping these cell clones under control, on the other side.

Nuclear membrane protein emerin: roles in gene regulation, actin dynamics and human disease.

Loss of emerin, a nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy (EDMD), characterized by muscle weakening, contractures of major tendons and potentially lethal cardiac conduction system defects. Emerin has a LEM-domain and therefore binds barrier-to-autointegration factor (BAF), a conserved chromatin protein essential for cell division. BAF recruits emerin to chromatin and regulates higher-order chromatin structure during nuclear assembly. Emerin also binds filaments formed by A-type lamins, mutations in which also cause EDMD. Other partners for emerin include nesprin-1alpha and transcriptional regulators such as germ cell-less (GCL). The binding affinities of these partners range from 4nM (nesprin-1alpha) to 200 nM (BAF), and are physiologically significant. Biochemical studies therefore provide a valid means to predict the properties of emerin-lamin complexes in vivo. Emerin and lamin A together form stable complexes with either BAF or GCL in vitro. BAF, however, competes with GCL for binding to emerin in vitro. These and additional partners, notably actin and nuclear myosin II, suggest disease-relevant roles for emerin in gene regulation and the mechanical interity of the nucleus.

The nuclear lamina comes of age.

Many nuclear proteins form lamin-dependent complexes, including LEM-domain proteins, nesprins and SUN-domain proteins. These complexes have roles in chromatin organization, gene regulation and signal transduction. Some link the nucleoskeleton to cytoskeletal structures, ensuring that the nucleus and centrosome assume appropriate intracellular positions. These complexes provide new insights into cell architecture, as well as a foundation for the understanding of the molecular mechanisms that underlie the human laminopathies - clinical disorders that range from Emery-Dreifuss muscular dystrophy to the accelerated ageing seen in Hutchinson-Gilford progeria syndrome.

X-linked form of Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an inherited muscular disorder clinically characterized by slowly progressive weakness affecting humero-peroneal muscles, early joint contractures and cardiomyopathy with conduction defects. Autosomal dominant and recessive forms are caused by mutations in lamin A/C gene. Lamin A/C is a major component of nuclear lamina, and its gene mutations cause several human disorders including muscular dystrophy, cardiomyopathy, lipodystrophy, neuropathy, and progeria syndrome. X-linked recessive form of EDMD is caused by mutation in EMD (or STA) gene encoding an integral protein of the inner nuclear membrane. Emerin expresses ubiquitously, but its deficiency affects only limited tissues of skeletal and cardiac muscles and joints. In this paper, I will focus on clinical and pathological aspects of X-EDMD and possible functions of emerin.

Multiple and surprising new functions for emerin, a nuclear membrane protein.

Emerin is an integral protein of the nuclear inner membrane. Emerin is not essential, but its loss of function causes Emery-Dreifuss muscular dystrophy. We summarize significant recent progress in understanding emerin, which was previously known to interact with barrier-to-autointegration factor and lamins. New partners include transcription repressors, an mRNA splicing regulator, a nuclear membrane protein named nesprin, nuclear myosin I and F-actin. These interactors imply multiple roles for emerin in the nucleus, some of which overlap with related LEM-domain proteins.

[Role of splenin and its active factor in regulating liver monooxygenase system in experimental immunodeficiency and hepatosohepatitis]. / Rol' spleninu ta ioho aktyvnoho chynnyka v rehuliatsii monoksyhenaznoi systemy pechinky za eksperimental'nykh imunodefshchytu ta hepatozohepatytu.

An influence of splenin and its non-peptide factor of splenin (NFS) on the state of cytochrome P-450 dependent monooxygenase system (MOS) of liver microsomes in healthy animals under immunodeficiency (splenectomy, administration of gamma-aminobutyric acid (GABA) and toxic hepatosohepatitis was studied. The stimulating action of splenin and NFS on cytochrome P-450 content and MOS activity of liver microsomes in healthy animals has been established. The indices studied markedly decreased after splenectomy. The splenin or NFS administrations promote the recovery of these indices up to starting level in asplenic animals. A decrease in thymic mass dependent in GABA administration is prevented by NFS pretreatment of animals; there is no any effect of mediator acid on cytochrome P-450 content and MOS activity was noted. The preliminary administration NFS potentiates hepatotoxic effect of carbon tetrachloride and increases its inhibitory effect on P-450 dependent MOS of liver microsomes. Under the NFS action the effect in activity of the last is caused by the factor influence on the reparative processes in the liver.

Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF.

Loss of emerin, a lamin-binding nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy. We analyzed 13 site-directed mutations, and four disease-causing mutations that do not disrupt emerin stability or localization. We show that emerin binds directly to barrier-to-autointegration factor (BAF), a DNA-bridging protein, and that this binding to BAF requires conserved residues in the LEM-motif of emerin. Emerin has two distinct functional domains: the LEM-domain at the N-terminus, which mediates binding to BAF, and a second functional domain in the central region, which mediates binding to lamin A. Disease mutation Delta95-99 mapped to the lamin-binding domain and disrupted lamin A binding in vitro. Two other disease-linked residues, Ser54 and Pro183, mapped outside the BAF and lamin-binding domains, suggesting that emerin may have additional functional domains relevant to disease. The disease-linked emerin proteins all remained active for binding to BAF, both in vitro and in vivo, suggesting that disease can result from the loss of specific molecular interactions between emerin and either lamin A or putative novel partner(s). The demonstration that emerin binds directly to BAF, coupled to similar results for LAP2, provides proof in principle that all LEM-domain nuclear proteins can interact with BAF, with interesting implications for chromatin attachment to the nuclear envelope.

Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes.

We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.

On the role of thymopoietins in cell proliferation. Immunochemical evidence for new members of the human thymopoietin family.

Thymopoietins (TMPOs) are a group of ubiquitously expressed nuclear proteins. They are suggested to play an important role in nuclear envelope organization and cell cycle control, as has been shown for lamina-associated polypeptides 2 alpha and beta, which are the rat homologs of human TMPOalpha and TMPObeta, respectively. The recent isolation and characterization of seven mouse TMPO mRNA transcripts named TMPO-alpha, beta, beta', gamma, epsilon delta and zeta, suggest that more than the three previously reported transcripts, alpha, beta, and gamma forms, may exist in humans. Here we report on the demonstration of putative human TMPOdelta and epsilon by immunoblotting of human cell lines using a newly prepared polyclonal antiserum against the common N-terminal region of TMPO. Furthermore, we prepared the first truly TMPO-beta-specific, affinity-purified polyclonal antiserum, using a part of the human analog of the beta-specific domain of mouse TMPO 220-259 for immunization. We showed that human TMPObeta is highly expressed in all cancerous cells tested, while hardly any cross-reactivities with other proteins could be detected. In contrast to the high expression of human TMPObeta in the cancer-derived neuroblastoma cell lines SK-N-MC and SMS-KAN, we found very low expression of human TMPObeta in low-proliferative nerve tissue. These data led us to the assumption that expression of TMPObeta may correlate with the occurrence of cancer, and therefore may serve as a new tumor marker, or even as a new target for cancer therapy.

Heart-specific localization of emerin: new insights into Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked inherited disease characterized by early contracture of the elbows, Achilles tendons and post-cervical muscles, slow progressive muscle wasting and weakness and cardiomyopathy presenting with arrhythmia and atrial paralysis: heart block can eventually lead to sudden death. The EDMD geneencodes a novel ubiquitous protein, emerin, which decorates the nuclear rim of many cell types. Amino acid sequence homology and cellular localization suggested that emerin is a member of the nuclear lamina-associated protein family. These findings did not explain the role of emerin nor account for the skeletal muscle- and heart-specific clinical manifestations associated with the disorder. Now we report that emerin localizes to the inner nuclear membrane, via its hydrophobic C-terminal domain, but that in heart and cultured cardiomyocytes it is also associated with the intercalated discs. We propose a general role for emerin in membrane anchorage to the cytoskeleton. In the nuclear envelope emerin plays a ubiquitous and dispensable role in association of the nuclear membrane with the lamina. In heart its specific localization to desmosomes and fasciae adherentes could account for the characteristic conduction defects described in patients.

Cytofluorometric and cytomorphologic analysis of human bone marrow cells derived from stromal cultures stimulated by granulocyte-macrophage colony-stimulating factor, interferon-gamma and splenopentin pentapeptide.

We studied the influence of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF), human recombinant interferon-gamma (hrIFN-gamma) and splenopentin pentapeptide (Sp-5), either alone or in combination, on the proliferation and differentiation of human bone marrow cells in modified Dexter's cultures. After 10, 14 and 21 days cells were analyzed by classical staining according to Pappenheim and by cytofluorometry with a set of different monoclonal antibodies. IFN-gamma inhibited the proliferation of progenitor cells and provided signals promoting monocytic differentiation, whereas GM-CSF induced the proliferation of blastoid elements which expressed HLA-DR and M2 (VIM-2 monoclonal antibody), but progressively lost surface CD34. Furthermore, an increase of CD15+ cells was also observed. When GM-CSF was tested in combination with IFN-gamma, it abolished the inhibitory effect of IFN-gamma and both cytokines synergized to promote the expression of CD11c, CD14 and M2 surface antigens. Sp-5 alone had only a marginal activity, but it potentiated the effects of GM-CSF. These findings suggest that GM-CSF may induce the transition from stem cells to committed myeloid progenitors. In contrast to IFN-gamma, Sp-5 can serve as an additional proliferative signal with negligible effects on cell maturation.

Thymopentin induces release of ACTH-like immunoreactivity by human lymphocytes.

Peripheral mononuclear (PMN) cells are known to produce ACTH-like immunoreactivity (ACTH-LIR) in vitro. Based on these findings the aim of this study was to find out whether thymopentin (the active pentapeptide of the native hormone thymopoietin) may stimulate ACTH-LIR production and release by cultured normal human lymphocytes. Thymopentin at concentration of 1 microgram/ml was capable of inducing ACTH-LIR release by normal human PMN cells (median 22 pg/ml) whereas ACTH-LIR inside cells was lower (median 11 pg/10(7) cells). The chromatographic characterization of the eluted material identified the presence of ACTH immunoreactive peptides with the elution characteristics of the precursors 31 K proopiomelanocortin, 22 K ACTH and 4.5 K ACTH, together with higher molecular weight material (greater than 43 K). These data demonstrate that thymopentin induces ACTH-LIR release by human lymphocytes, thus adding a novel factor to those already reported (corticotrophin releasing factor, lipopolysaccharide, viruses) capable of such function.