Continuous regulation is required to maintain a given cell state or to allow it to change in response to the environment. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes, the tetracycline (tet) regulatable system has distinct advantages. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.
In multinucleated heterokaryons formed from the fusion of differentiated muscle cells to either hepatocytes or fibroblasts, muscle-specific gene expression is activated, liver-specific gene expression is repressed, and there are changes in the location of the Golgi apparatus. An understanding of the regulatory mechanisms that underlie this plasticity is of particular interest given the stability of the differentiated state in vivo. We have now investigated whether MyoD or myogenin, regulators of muscle-specific gene expression that have a helix-loop-helix motif, can induce the phenotypic conversion observed in heterokaryons. When these regulators were stably or transiently introduced into fibroblasts or hepatocytes by microinjection, transfection or retroviral infection with complementary DNA in expression vectors, fibroblasts expressed muscle-specific genes, whereas hepatocytes did not. However, fusion of hepatocytes stably expressing MyoD to fibroblasts resulted in activation in the heterokaryon of muscle-specific genes of both cell types. These results imply that other regulators, present in fibroblasts but not in hepatocytes, are necessary for the activation of muscle-specific genes, and indicate that the differentiated state of a cell is dictated by its history and a dynamic interaction among the proteins that it contains.
We present a method for monitoring receptor dimerization at the membrane of live cells. Chimeric proteins containing the epidermal growth factor (EGF) receptor extracellular and transmembrane domains fused to weakly complementing beta-galactosidase (beta-gal) deletion mutants were expressed in cells in culture. Treatment of the cells with EGF-like compounds for as little as 15 s resulted in chimeric receptor dimerization detectable as beta-gal enzymatic activity. The dose response of chimeric receptors was ligand specific. beta-galactosidase complementation was reversible upon removal of ligand and could be reinduced. Antibodies that block ligand binding inhibited receptor dimerization and beta-gal complementation. These results demonstrate that beta-gal complementation provides a rapid, simple, and sensitive assay for protein interactions and for detecting and monitoring the kinetics of receptor dimerization.
Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.Myasthenia gravis (MG) is a human autoimmune disorder that affects neuromuscular function by impairing synaptic transmission. MG patients have circulating autoantibodies directed against nicotinic acetylcholine receptors (AChRs) concentrated on skeletal muscle at neuromuscular junctions (1-3). These antibodies reduce the sensitivity of muscle to acetylcholine by blocking ligand binding sites and by increasing the rate of AChR degradation (4-10). Glucocorticoids used in conjunction with acetylcholinesterase (AChE) inhibitors are currently among the most effective pharmacologic treatments of the debilitating clinical symptoms of MG and may be responsible for the 50% reduction in mortality in the past two decades (11). The recovery of motor strength after glucocorticoid therapy is thought to result from the welldocumented immunosuppressive effects of steroids (12, 13). However, the titer of anti-AChR antibodies in MG patients treated with steroids often does not correlate with the severity of the disease or incidence of death (11, 14). We demonstrate here that the glucocorticoid dexamethasone (Dex) directly affects cultured human myotubes by inducing AChR synthesis and increasing total surface AChRs 2-fold. In contrast, Dex has no effect on other parameters of muscle differentiation such as fusion index, AChE activity, and sarcomeric actin transcript accumulation. The effect of Dex on AChR levels is evident in the presence of sera from MG patients, indicating that the therapeutic benefits of corticosteroid therapy are likely to derive from effects not only on the immune system but also on muscle tissue. These results suggest that therapeutic strategies directed at promoting tissue-specific gene expression will prove useful in MG as well as other human genetic and autoimmune disorders characterized by reduced levels of proteins including cell surface receptors. MATERIALS AND METHODSMuscle Cultures. Individual muscle clones were isolated as described (15) from the vastus lateralis muscle of three individuals (XXVI, XXXII, and XXVIII aged 2, 7, and 63 years, respect...
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