In vitro system for differentiating pluripotent neural crest cells into smooth muscle cells

The change in vascular smooth muscle cells (SMC) from a differentiated to a dedifferentiated state is the critical phenotypic response that promotes occlusive arteriosclerotic disease. Despite its importance, research into molecular mechanisms regulating smooth muscle differentiation has been hindered by the lack of an in vitro cell differentiation system. We identified culture conditions that promote efficient differentiation of Monc-1 pluripotent neural crest cells into SMC. Exclusive Monc-1 to SMC differentiation was indicated by cellular morphology and time-dependent induction of the SMC markers smooth muscle alpha-actin, smooth muscle myosin heavy chain, calponin, SM22alpha, and APEG-1. The activity of the SM22alpha promoter was low in Monc-1 cells. Differentiation of these cells into SMC caused a 20-30-fold increase in the activity of the wild-type SM22alpha promoter and that of a hybrid promoter containing three copies of the CArG element. By gel mobility shift analysis, we identified new DNA-protein complexes in nuclear extracts prepared from differentiated Monc-1 cells. One of the new complexes contained serum response factor. This Monc-1 to SMC model should facilitate the identification of nodal regulators of smooth muscle development and differentiation.     

Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.     

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)-derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4 degrees C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 microg/ml. In experiments at 37 degrees C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole-hexanoic acid-BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1-50 microg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMC migration was chemotactic in nature and was significantly inhibited (approximately 80%) by an antibody against transforming growth factor-beta (TGF-beta), and the amount of TGF-beta secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-beta is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.