Vascular injury involves phenotypic changes in smooth muscle cells (SMCs) of the arterial wall, including increased proliferation potential, changes in lipid metabolism, greater extracellular matrix production, and decreased expression of several structural and contractile proteins. To understand how injury decreases contractile protein synthesis, we must first know how SMCs control their synthesis normally. Our laboratory has focused on transcriptional control at the smooth muscle alpha actin (SMalphaA) promoter, both to understand contractile protein regulation, and as a significant component of phenotypic modulation by SMCs. Although other cells also express SMalphaA, there is smooth muscle- specific transcriptional regulation of this promoter, which depends on multiple cis and trans elements (1). Our published experiments found the first 125bp 5' to the start site in the rat SMalphaA promoter were transcriptionally active in multiple cell lines, and that cell-type specific expression required regulator elements lying -125 to -547bp upstream (1). This region includes 3 potential E-box elements (E1, E2, and E3), which are binding sites for basic helix-loop-helix (bHLH) transcriptional factors. Basic HLH homo- and heterodimers play a key role in coordinate control of gene expression during differentiation in skeletal muscle and other tissues. The central focus of this proposal is to determine whether bHLH heterodimers also bind to E-boxes in the SMalphaA promoter. In preliminary experiments, I compared the activity of native and mutated -271bp promoters using CAT reporter assays. Constructs with mutations that disrupted either the E1 or E2 site alone were fully active in SMCs, while double mutations of both E1 and E2 sites abolished activity. In contrast, L6 skeletal myotubes required both E1 and E2 sites for CAT activity. In electrophoretic mobility shift assays (EMSA), smooth muscle and L6 myotube nuclear proteins also bound to DNA oligo probes containing either the native E1 or E2 sites, but not to probes with the same inactivating mutations as CAT assays. The central hypothesis of this proposal is that transcription of the SMalphaA gene requires a single E-box which binds bHLH protein heterodimers, and that one member of the dimer is ubiquitous while the second is SMC restricted. This hypothesis will be tested by addressing two aims.
AIM #1 will utilize site-directed mutagenesis and transient transfection assays to characterize the nucleotides within the E1 and E2 sites that are required for transcriptional activity of the -271bp SMalphaA promoter in rat aortic SMCs. Comparison of required nucleotides to the conserved E-box motif, 5'-CAnnTG-3', will determine whether E1 or E2 are functioning as true E-box elements. One to 4bp mutations in both of the putative E-box sites will assayed using CAT reporters, to determine minimal nucleotides required for promoter transcriptional activity. Results of methylation footprinting (MFP) will identify specific nucleotides in or near E1 and E2 sites that are required for SMC nuclear protein binding, and whether they are the same nucleotides that are functionally important in CAT reporter studies.
AIM #2 will identify SMC proteins that bind at E1 or E2 sites to regulate transcription by the SMalphaA promoter.
Aim #2 A will test the hypothesis that the SMalphaA E1 and E2 binding complexes consist of a heterodimer of a known Type I bHLH factor with a smooth muscle restricted or selective bHLH partner. Known bHLH proteins will be identified by EMSA supershifting assays using bHLH-1-specific antibodies, by DNA crosslinking to estimate molecular size, and by comparison of methylation footprints on E1 and E2 by SMCs to footprints of known bHLH-1 proteins. Additional comparisons to E1 and E2 footprints make by rat endothelial cell (ECs), skeletal myoblast and myotube, and fibroblast nuclear proteins will determine whether a smooth muscle specific protein is present in E1 or E2 binding complexes.
Aim #2 B will be to clone potentially novel smooth muscle-restricted bHLH factors that bind E1 and E2 sites, thereby regulating transcriptional control of SMalphaA promoter. The cloning strategy selected will depend upon data from Aim #2A, but may include the yeast two-hybrid system, using bHLH-1 proteins identified in Aim #2A as """"""""bait."""""""" The proposed studies will increase our understanding of tissue-specific regulation by E-box enhancer motifs, by determining whether the bHLH family of transcription factors bind to SMalphaA promoter in SMCs. These data will also enhance our understanding to molecular regulation of SMC differentiation at the level of gene transcription, and lead to further investigations of bHLH-mediated pathways which alter SMalphaA expression during vascular disease. Finally, this study complements my long-term interest in the acute versus stable changes in phenotype that occur in smooth muscle cells in response to injury.
| Swartz, E A; Johnson, A D; Owens, G K (1998) Two MCAT elements of the SM alpha-actin promoter function differentially in SM vs. non-SM cells. Am J Physiol 275:C608-18 |
