Adipocyte differentiation involves the transcriptional activation of several genes in triglyceride metabolism, including the adipose P2 (aP2 or 422) gene that encodes the adipocyte lipid-binding protein ALBP. Within the mouse aP2 promoter region, the AE-1 sequence functions as either a positive or a negative element in the regulation of aP2 gene expression. The AE-1 sequence is the binding site for the positive murine (3T3) adipocyte factor C/EBP-alpha, several human preadipocyte factors, and a 3T3 preadipocyte factor(s) that has been implicated as a repressor of aP2 gene expression. Here we report the cloning of new complementary DNAs that encode the 3T3 preadipocyte factor (termed AEBP1) and demonstrate that AEBP1 expression is abolished during adipocyte differentiation. Furthermore, we show that an activity of a carboxypeptidase associated with AEBP1 is important in the transcriptional repression function of AEBP1. Thus AEBP1 might represent a new type of transcription factor that regulates transcription by cleavage of factors involved in transcription.
Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder characterized by immune-mediated inflammation of the extraocular muscles and orbital connective tissue. TAO is linked, in a unique way, with thyroid autoimmunity, in particular Graves' hyperthyroidism. Our working hypothesis for the pathogenesis of TAO is that recognition of a thyrotropin receptor (TSHR)-like protein in the orbital preadipocytes by antibodies may be the initial event leading to homing of lymphocytes into the orbital tissues. In the course of thyroid inflammation, antibodies and T cells reactive against G2s expressed in thyroid membranes cross-react with the protein in the eye muscle fiber, leading to eye muscle damage and dysfunction. Those patients with anti-G2s antibodies develop ocular myopathy. Antibodies against flavoprotein, the 64-kDa protein, which are produced in the context of eye muscle fiber damage and mitochondrial rupture, are sensitive markers of immune-mediated fiber necrosis in patients with ophthalmopathy but do not directly damage the eye muscle. Antibodies against type XIII collagen, which is localized in the plasma membranes of orbital fibroblast, may be a new marker for the congestive ophthalmopathy subtype of TAO. The measurement of antibodies against key eye muscle and orbital connective tissue autoantigens may have a role in the management of active ophthalmopathy and its prediction in patients with Graves' hyperthyroidism.
The eye changes associated with Graves' hyperthyroidism can be classified into two subtypes, congestive ophthalmopathy (CO), in which inflammatory changes in the periorbital tissues predominate, and ocular myopathy (OM), in which eye muscle damage is the main feature. Antibodies against the flavoprotein (Fp) subunit of succinate dehydrogenase (SDH), the 64-kd protein, and G2s, a thyroid and eye muscle shared protein of unknown function, are good markers of eye muscle cell damage in patients with OM. Another antigen associated with ophthalmopathy is the flavine adenine nucleotide (FAD) cofactor of several mitochondrial enzymes, including SDH. We tested for serum antibodies against purified human recombinant Fp, FAD, and a G2s fusion protein, in patients with thyroid-associated ophthalmopathy (TAO) and control patients and subjects, in enzyme-linked immunosorbent assay. Antibodies against Fp were detected in 32% of patients with TAO, 30% with Graves' hyperthyroidism (GH), 16% with Hashimoto's thyroiditis (HT), in 14% of patients with multi-nodular goiter (MNG), and in 6% of normal subjects. Antibodies against FAD were found in 24%, 30%, 24%, and 14%, respectively, of these patients and in 12% of the normals, while antibodies against G2s were detected in 50% of patients with TAO, 40% with GH, 40% with HT, in 29% of patients with MNG, and in 7% of normals. We also tested for antibodies against SDH, FAD, and G2s in 12 patients with GH who developed CO (6 patients) or OM (6 patients) after treatment with antithyroid drugs. Of the 6 patients who developed OM, antibodies against SDH preceded the onset of eye disease in 4 and coincided with it in 2, antibodies against G2s preceded eye muscle disease in 5 and coincided with it in 1 patient while antibodies against FAD preceded the development of OM in 5 patients. Of the 6 patients who developed CO, antibodies against SDH were detected in only one patient and borderline levels were demonstrated in 1, while anti-FAD and anti-G2s each preceded the onset of eye signs in 6 patients. Positive sera from another group of patients with TAO, and a second group of normal subjects, were tested at increasing serum dilutions. Sera from the two groups showed similar dilution patterns, except for a few patients with TAO in whom increasing dilutions was associated with increased, then decreased, antibody levels. In this experiment the prevalences of the two antibodies were much greater in patients with TAO namely, 67% for anti-Fp and 89% for anti-G2s, while the prevalences in the normals were 11% and 22%, respectively. The reason for this apparent discrepancy is not clear but may reflect subject and assay differences. Because Fp is found within the mitochondrial membrane it is likely that the corresponding antibodies are produced after eye muscle necrosis, and do not play a role in its pathogenesis. The primary reaction in the eye muscle may be T-cell autoimmunity against G2s, although this has not been proven. The mechanism for the production of antibodies against G2s, FAD, and Fp in subj...
Serum autoantibodies against eye muscle antigens are closely linked with thyroid-associated ophthalmopathy (TAO), although their significance is unclear. The two antigens that are most often recognized are eye muscle membrane proteins with molecular masses of 55 and 64 kDa, as determined from immunoblotting with crude human or porcine eye muscle membranes. We cloned a fragment of the 55-kDa protein by screening an eye muscle expression library with affinity-purified anti-55 kDa protein antibody prepared from a TAO patient's serum. A complementary DNA (cDNA) encoding a novel protein, which we have called G2s, was sequenced on both strands, and its size was 411 bp. The open reading frame of G2s corresponded to a 121-amino acid peptide with a size of 1.4 kb. Using the rapid amplification of 5'-cDNA ends technique we were able to clone an additional 0.3 kb of the protein. G2s did not share significant homologies with any other entered protein in computer databases and had one putative transmembrane domain. Using the 1.4 kb cDNA as probe in Northern blotting of a panel of messenger ribonucleic acids prepared from human tissues, the parent protein was shown to correspond to a large molecule of about 5.8 kb with a calculated molecular mass of approximately 220 kDa, consistent with earlier immunoblot studies performed in the absence of reducing agents. G2s was strongly expressed in eye muscle, thyroid, and other skeletal muscle and to a lesser extent in pancreas, liver, lung, and heart muscle, but not in kidney or orbital fibroblasts. We tested sera from patients with Graves' hyperthyroidism with and without ophthalmopathy and from control patients and subjects for antibodies against a G2s fusion protein by immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting, antibodies reactive with G2s were identified in 70% of patients with TAO of less than 3 yr duration, 53% with TAO of more than 3 yr duration, 36% with Graves' hyperthyroidism without evident ophthalmopathy, 17% with Hashimoto's thyroiditis, 3% with type 1 diabetes, 23% with nonimmunological thyroid disorders, and 16% of normal subjects. The prevalences, compared to normal values, were significant for the two groups of patients with TAO, but not for the other groups. Tests were positive in 54% of patients with active TAO, 33% with chronic ophthalmopathy, 36% with Graves' hyperthyroidism, 54% with Hashimoto's thyroiditis, 23% with type 1 diabetes, and in 11% of normal subjects using enzyme-linked immunosorbent assay. The antibodies predicted the development of the ocular myopathy subtype of TAO in six of seven patients and the congestive ophthalmopathy subtype in seven of eight patients, respectively, with Graves' hyperthyroidism studied prospectively during and after antithyroid drug therapy. Antibodies reactive with G2s may be early markers of ophthalmopathy in patients with Graves' hyperthyroidism. Because G2s is expressed in both thyroid and eye muscle, immunoreactivity against a shared epitope in the two tissues may explain the well known link...
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