No abstract

Muscular dystrophy is a heterogeneous genetic disease that affects skeletal and cardiac muscle. The genetic defects associated with muscular dystrophy include mutations in dystrophin and its associated glycoproteins, the sarcoglycans. Furthermore, defects in dystrophin have been shown to cause a disruption of the normal expression and localization of the sarcoglycan complex. Thus, abnormalities of sarcoglycan are a common molecular feature in a number of dystrophies. By combining biochemistry, molecular cell biology, and human and mouse genetics, a growing understanding of the sarcoglycan complex is emerging. Sarcoglycan appears to be an important, independent mediator of dystrophic pathology in both skeletal muscle and heart. The absence of sarcoglycan leads to alterations of membrane permeability and apoptosis, two shared features of a number of dystrophies. β‐sarcoglycan and δ‐sarcoglycan may form the core of the sarcoglycan subcomplex with α‐ and γ‐sarcoglycan less tightly associated to this core. The relationship of ϵ‐sarcoglycan to the dystrophin‐glycoprotein complex remains unclear. Animals lacking α‐, γ‐ and δ‐sarcoglycan have been described and provide excellent opportunities for further investigation of the function of sarcoglycan. Dystrophin with dystroglycan and laminin may be a mechanical link between the actin cytoskeleton and the extracellular matrix. By positioning itself in close proximity to dystrophin and dystroglycan, sarcoglycan may function to couple mechanical and chemical signals in striated muscle. Sarcoglycan may be an independent signaling or regulatory module whose position in the membrane is determined by dystrophin but whose function is carried out independent of the dystrophin‐dystroglycan‐laminin axis. Microsc. Res. Tech. 48:167–180, 2000. © 2000 Wiley‐Liss, Inc.

Recent data indicate that sterol carrier protein-2 (SCP-2) functions in the rapid movement of newly synthesized cholesterol to the plasma membrane (Puglielli, L., Rigotti, A., Greco, A. V., Santos, M. J., and Nervi, F. (1995) J. Biol. Chem. 270, 18723-18726). In order to further characterize the cellular function of SCP-2, we transfected McA-RH7777 rat hepatoma cells with a pre-SCP-2 cDNA expression construct. In stable transfectants, pre-SCP-2 processing resulted in an 8-fold increase in peroxisomal levels of SCP-2. SCP-2 overexpression increased the rates of newly synthesized cholesterol transfer to the plasma membrane and plasma membrane cholesterol internalization by 4-fold. There was no effect of SCP-2 overexpression on the microsomal levels of acyl-CoA:cholesterol acyltransferase and neutral cholesterol ester (CE) hydrolase; however, in the intact cell, CE synthesis and mass were reduced by 50%. SCP-2 overexpression also reduced high density lipoprotein-cholesterol secretion and apoA-I gene expression by 70% and doubled the rate of plasma membrane desmosterol conversion to cholesterol. We conclude that SCP-2 overexpression enhances the rate of cholesterol cycling, which reduces the availability of cholesterol for CE synthesis and alters the activity of a cellular cholesterol pool involved in regulating apoA-Imediated high density lipoprotein cholesterol secretion. The net result of these changes in cholesterol metabolism is a 46% increase in plasma membrane cholesterol content, the implications of which are discussed. Cellular free cholesterol is predominantly located in the plasma membrane (reviewed in Ref. 1). Cellular cholesterol content, however, is determined by the concerted action of intracellular enzymes and regulatory proteins as follows: ACAT 1 which catalyzes the synthesis of CE, sterol regulatory element binding proteins, which regulate the transcription of a number of genes involved in cholesterol metabolism (2, 3), and various cell type-specific metabolic reactions, e.g. lipoprotein secretion, steroidogenesis, and bile acid synthesis. Since cholesterol is highly insoluble in an aqueous environment, it has been postulated that sterol carrier protein-2 (SCP-2) regulates the movement and thus the availability of cholesterol for different cellular processes (4 -6). The evidence supporting this contention was initially derived in large part from studies demonstrating that the addition of purified SCP-2 stimulated the in vitro conversion of sterol intermediates to cholesterol (7) and cholesterol to 7␣-hydroxycholesterol (8) steroid hormones (9 -13) and cholesterol ester (14). More recent studies indicate that SCP-2 gene expression is regulated by changes in cellular cholesterol content (15-17); however, in these studies, a direct role in cholesterol trafficking and esterification was not demonstrated. The strongest support for a role in cellular cholesterol metabolism comes from studies on the role of SCP-2 in steroidogenesis. SCP-2 gene expression is coordinately regulated with steroid hormone syn...

The sarcoglycan complex is found normally at the plasma membrane of muscle. Disruption of the sarcoglycan complex, through primary gene mutations in dystrophin or sarcoglycan subunits, produces membrane instability and muscular dystrophy. Restoration of the sarcoglycan complex at the plasma membrane requires reintroduction of the mutant sarcoglycan subunit in a manner that will permit normal assembly of the entire sarcoglycan complex. To study sarcoglycan gene replacement, we introduced transgenes expressing murine ␥-sarcoglycan into muscle of normal mice. Mice expressing high levels of ␥-sarcoglycan, under the control of the muscle-specific creatine kinase promoter, developed a severe muscular dystrophy with greatly reduced muscle mass and early lethality. Marked ␥-sarcoglycan overexpression produced cytoplasmic aggregates that interfered with normal membrane targeting of ␥-sarcoglycan. Overexpression of ␥-sarcoglycan lead to the up-regulation of ␣-and ␤-sarcoglycan. These data suggest that increased ␥-sarcoglycan and/or mislocalization of ␥-sarcoglycan to the cytoplasm is sufficient to induce muscle damage and provides a new model of muscular dystrophy that highlights the importance of this protein in the assembly, function, and downstream signaling of the sarcoglycan complex. Most importantly, gene dosage and promoter strength should be given serious consideration in replacement gene therapy to ensure safety in human clinical trials.