| Phenotypic switching of smooth muscle cells (SMCs) to a less differentiated state is known to contribute to many vascular diseases including atherosclerosis, hypertension, and post-revascularization stenosis. However, the mechanisms that control SMC gene expression during normal development/maturation and in response to environmental cues that elicit phenotypic switching remain largely undefined. The CC(A/T)6GG (CArG)/serum response factor (SRF) interaction is required for transcriptional activation of numerous smooth, skeletal, and cardiac muscle-specific genes as well as many growth response genes such as c-fos. The focus of this dissertation project was to elucidate the mechanisms that regulate CArG/SRF-dependent SM-specific gene expression.; Studies in transgenic mice revealed that the highly conserved degeneracy and resultant reduced SRF binding affinity of the SM alpha-actin 5 ' CArG elements, as compared to consensus CArGs, is not required for cell-specific control of SM alpha-actin gene expression during normal development/maturation in vivo. These studies are inconsistent with cell culture studies, which showed that substitution of a c-fos consensus CArG for one or both 5' alpha-actin CArGs resulted in relaxed cell specificity of SM alpha-actin gene expression. Of major significance, these substitutions significantly attenuated downregulation of SM alpha-actin in response to vascular injury in vivo. Further studies using chromatin immunoprecipitation (ChIP) assays and real time RT-PCR indicated that loss of the potent SRF co-factor, myocardin, in combination with the degenerate CArG elements within the promoter, may contribute to selective repression of SM alpha-actin in response to vascular injury and play a key role in phenotypic switching of SMCs. Additional studies showed that the ATTA homeodomain protein binding motif in the SM alpha-actin promoter is required for maximal gene expression in vivo, thereby implicating the homeodomain family of proteins in control of CArG/SRF-dependent SM alpha-actin gene transcription.; With these studies in mind, we propose a model in which cell-specific regulation of SM alpha-actin is controlled by cooperative interactions between multiple cis-regulatory elements, including the degenerate CArGs and the ATTA motif, and trans-acting factors, including SRF, myocardin, and as yet undefined homeodomain proteins, in a manner unique to and determined by the local environmental cues within the SMC at any particular time. |
