Induction of stearoyl CoA desaturase is associated with high-level induction of emerin RNA.

The genes encoding enzymes involved in fatty acid metabolism are regulated by sterols. Stearoyl CoA desaturase, a key enzyme in the synthesis of unsaturated fatty acyl-CoAs is transcriptionally regulated by fat-free diet and sterols. To identify other genes that are induced in rat liver by fat-free diet we performed an in vivo gene expression profile analysis using DNA microarrays. Here we report that among the genes highly expressed is emerin, an integral protein of the inner nuclear membrane. Mutated or nonexpressed emerin occurs in patients with muscular dystrophy. Sterol regulatory element binding proteins activate the transcription of several sterol regulated genes. To investigate whether sterol regulatory element binding proteins or indirectly cholesterol activates the transcription of stearoyl CoA desaturase and emerin, we cultured Chinese hamster ovary (CHO), either in cholesterol-rich or cholesterol-depleted mediums. We also transiently transfected the cell culture with plasmid encoding sterol regulatory element binding proteins in cholesterol-rich medium. Our data show that cholesterol-supplemented media as well as the transient transfection induced the expression of stearoyl CoA desaturase RNA 3.5- and 7-fold respectively. However, the RNA level of emerin was not altered under these conditions, implying that the parallel induction of emerin is independent of the sterol regulatory element binding regulation pathway.

Prenatal undernutrition and postnatal growth are associated with adolescent thymic function.

The fetal and early infant origins of a number of adult cardiovascular and metabolic diseases have received considerable attention, but the long-term consequences of early environments for human immune function have not been reported. We investigated the effects of pre- and postnatal environments on thymic hormone production in adolescents participating in an ongoing longitudinal study in the Philippines. Prospective data collected at birth, during y 1 of life, in childhood and in adolescence were used to predict plasma thymopoietin concentration in 14- to 15-y-old adolescents (n = 103). Thymopoietin concentration was compared for small-for-gestational-age and appropriate-for-gestational-age individuals while controlling for a range of postnatal exposures. Prenatal undernutrition was significantly associated with reduced thymopoietin production in interaction with the duration of exclusive breast-feeding (P = 0.006). Growth in length during y 1 of life was positively associated with adolescent thymopoietin production (P = 0.002). These associations remained significant after adjusting for a range of potentially confounding variables. These findings provide support for the importance of fetal and early infant programming of thymic function, and suggest that early environments may have long-term implications for immunocompetence and adult disease risk.

Emerin expression at the early stages of myogenic differentiation.

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.

Mutations in Emery-Dreifuss muscular dystrophy and their effects on emerin protein expression.

Seventeen families with Emery-Dreifuss muscular dystrophy (EDMD) have been studied both by DNA sequencing and by emerin protein expression. Fourteen had mutations in the X-linked emerin gene, while three showed evidence of autosomal inheritance. Twelve of the 14 emerin mutations caused early termination of translation. An in-frame deletion of six amino acids from the C-terminal transmembrane helix caused almost complete absence of emerin from muscle with no localization to the nuclear membrane, although mRNA levels were normal. This shows that mutant emerin proteins are unstable if they are unable to integrate into a membrane. A 22 bp deletion in the promoter region was expected to result in reduced emerin production, but normal amounts of emerin of normal size were found in leucocytes and lymphoblastoid cell lines. This shows that DNA analysis is necessary to exclude emerin mutations in suspected X-linked EDMD. Emerin levels in female carriers often deviated from the expected 50% and this was due, in at least two families, to skewed emerin mRNA expression from the normal and mutated alleles. In one family with a novel deletion of the last three exons of the emerin gene, a carrier had a cardiomyopathy and very low emerin levels (<5% of normal) due to skewed X-inactivation. In the three autosomal cases of EDMD, emerin was normal on western blots of blood cells, which suggests that autosomal EDMD is not caused by indirect reduction of emerin levels.

Reviewing the potential utility of interleukin-7 as a promoter of thymopoiesis and immune recovery.

The identification of interleukin-7 (IL-7) as a critical cytokine in early B- and T-cell development, combined with the discovery that it acts on mature T cells, opens new avenues for investigating the thymopoietic machinery and manipulation of the immune system. Initially, IL-7 was thought to be a growth factor in the context of the B-cell lineage in that it stimulates proliferation of early B-cell progenitors. However, it appears that this cytokine has a much broader field of activity within the network of signal transduction. Indeed, evidence exists to support the pivotal involvement of IL-7 in the gene rearrangement of the T-cell receptor repertoire that ultimately leads to thymocyte commitment. The finding that IL-7 is an inducer of both cytotoxic T-cell- and lymphocyte-activated killer cells is one of the significant recent developments in the field of tumor immunology. Lately, it has been demonstrated that administration of IL-7 to mice after myeloablative treatment accelerates immune recovery via a unique pathway. This review of the literature dealing with IL-7 in the realm of immune function shows, inter alia, the value of the cytokine in immunosuppressed animals. The collection of findings noted in this paper may be considered the forerunner for clinical application of IL-7 in a variety of conditions of hematolymphopoietic failure.

X-linked Emery-Dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

Six novel mutations in the emerin gene causing X-linked Emery-Dreifuss muscular dystrophy.

Mutations in the emerin gene, also referred to as the STA- or EMD-gene, have been found to be the cause of X-linked Emery-Dreifuss muscular dystrophy (EMD). For the present study an optimized set of primers was designed to amplify and sequence each of the six emerin gene exons, including the intron/exon boundaries. All emerin gene exons of 30 unrelated EMD patients have been screened by heteroduplex analysis. Aberrant patterns of single exons were found in seven patients. Direct sequencing of the respective exons revealed six novel mutations distributed in the promotor region and exons 3-6 (delta nt -19 to -40; delta AG nt 620-621; ins A nt 895; delta AT nt 908-909; C-->A nt 1420; ins TA nt 1570). By this study, the first mutations in the promotor region and in exon 5 have been identified. Each of the 25 mutations that have been described so far, including those from the present study, abolishes the synthesis of functional emerin. The mutations were submitted to the EMD Mutation database (http://www.path.cam.ac.uk/emd).

Demonstration of thymopoietin transcripts in different hematopoietic cell lines.

We have cloned and characterized the human thymopoietin (TP) coding region and studied the mRNA expression of this gene in different hematopoietic cell lines. The 150-bp PCR fragment that encodes the 49-amino-acid human TP peptide was isolated from genomic placental DNA. Its colinearity with the cDNA sequence suggests lack of introns within the coding region. TP mRNA expression was demonstrated in lymphocytes from all the differentiation stages investigated, as well as in a myeloid cell line (K-562). These findings suggest a further expansion of the proposed TP functions.

Three distinct human thymopoietins are derived from alternatively spliced mRNAs.

Thymopoietin (TP) was originally isolated as a 5-kDa 49-aa protein from bovine thymus in studies of the effects of thymic extracts on neuromuscular transmission and was subsequently observed to affect T-cell differentiation and function. We now report the isolation of cDNA clones for three alternatively spliced mRNAs that encode three distinct human T-cell TPs. Proteins encoded by these mRNAs, which we have named TP alpha (75 kDa), TP beta (51 kDa), and TP gamma (39 kDa), contain identical N-terminal regions, including sequences nearly identical to that of the originally isolated 49-aa protein, but divergent C-terminal regions. TP mRNAs are expressed in many tissues, most abundantly in adult thymus and fetal liver of the tissues so far examined. Distinct structural domains and functional motifs in TPs alpha, beta, and gamma suggest that the proteins have unique functions and may be directed to distinct subcellular compartments.