Expression of the SM22alpha promoter in transgenic mice provides evidence for distinct transcriptional regulatory programs in vascular and visceral smooth muscle cells

Parts used

Key findings

• The 445-bp proximal promoter of SM22α directed lacZ transgene expression in vascular smooth, cardiac, and skeletal muscle lineages of transgenic mouse embryos, recapitulating the temporospatial pattern of endogenous SM22α. Expression was first detected in the primitive heart tube at E8.75, expanded to the dorsal aorta by E9.0, and progressively marked all major conductive arterial vessels through E14.5.
• The SM22α-lacZ transgene was selectively active in conductive arterial but not venous or visceral smooth muscle cells, despite endogenous SM22α expression in all SMC subtypes. No transgene expression was detected in the inferior vena cava, femoral vein, or visceral SMCs of the gut, stomach, bladder, and bronchi at E13.5–E14.5, revealing that distinct transcriptional regulatory programs control gene expression across SMC lineages.
• Deletion analysis identified sequences between -445 and -144 bp as essential for SM22α promoter activity, with deletion to -144 bp reducing luciferase activity approximately sixfold in primary rat aortic SMCs. The promoter was threefold more active in aortic SMCs than in 10T1/2 fibroblasts and inactive in F9 teratocarcinoma cells. Two CArG boxes at -150 and -274 bp upstream of the transcription initiation site contributed to SMC-specific transcriptional regulation.

Abstract

SM22alpha is a putative calcium-binding protein that is expressed in cardiac, smooth, and skeletal muscle lineages during mouse embryogenesis and in adult smooth muscle cells (SMC). To define the mechanisms that regulate smooth muscle-specific gene transcription, we isolated the SM22alpha gene and analyzed its 5'-flanking region for elements that direct smooth muscle expression in transgenic mice. Using a series of promoter deletions, a region of the SM22alpha promoter containing 445 base pairs of 5'-flanking sequence was found to be sufficient to direct the specific expression of a lacZ transgene in mouse embryos in the vascular smooth, cardiac, and skeletal muscle lineages in a temporospatial pattern similar to the endogenous SM22alpha gene. However, in contrast to the endogenous gene, transgene expression was not detected in venous, nor visceral SMCs. This SM22alpha-lacZ transgene was therefore able to distinguish between the transcriptional regulatory programs that control gene expression in vascular and visceral SMCs and revealed heretofore unrecognized differences between these SMC types. These results suggest that distinct transcriptional regulation programs control muscle gene expression in vascular and visceral SMCs.