Identification of a 94-bp GC-rich element in the smooth muscle myosin heavy-chain promoter controlling vascular smooth muscle cell-specific gene expression.
The previously described rabbit 2.3-kilobase smooth muscle myosin heavy-chain (SMHCwt) promoter targets gene expression in transgenic animals to vascular smooth muscle cells (SMCs), including coronary arteries. Therefore, SMHCwt is thought to provide a promising tool for human gene therapy. In the present study, we examined tissue specificity and expression levels of wild-type and mutated SMHC promoters within the system of high-capacity adenoviral (hcAd) vectors. SMHCwt and a series of SMHC promoter deletion mutants, a triple promoter as well as a cytomegalovirus-SMHC hybrid promoter driving the enhanced green fluorescence protein (EGFP) reporter gene were transiently transfected into aortic SMCs. Fluorescence intensity was measured by flow cytometric analysis. Consecutively, hcAd vectors were constructed with the SMHCwt and the mutant promoter with the highest fluorescence activity. Levels of EGFP expression were determined after transduction of SMCs derived from human coronary arteries. For analysis of tissue specificity, embryonic stem (ES) cell-derived SMCs (ESdSMHCs) and cardiomyocytes (ESdCMs) were used. In comparison with SMHCwt, only the SMHCdel94 mutant lacking a 94-bp GC-rich element revealed a 1.5-fold increased fluorescence activity. Transduction of primary SMCs of human coronary arteries with hcAd vectors confirmed an increased EGFP expression driven by the SMHCdel94 promoter. In ES-cell-derived embryoid bodies, SMHCwt was exclusively active in transduced ESdSMCs. In contrast, expression of SMHCdel94 was also found in ESdCMs and other nontarget cells of the embryoid body. The tissue-specific rabbit SMHCwt promoter seems to be suitable for adenoviral gene transfer in SMCs of human coronary arteries and deletion of a 94-bp negative cis-acting GC-rich element results in loss of specificity.
Deindl
Elisabeth
E
Medical Clinic I, Department of Cardiology, Ludwig-Maximilians-University, 81377 Munich, Germany.
Middeler
Guido
G
Müller
Oliver J
OJ
Selbert
Stefan
S
Schlenke
Peter
P
Marienfeld
Uta
U
Thirion
Christian
C
Katus
Hugo A
HA
Franz
Wolfgang M
WM
D000818:Animals; D001482:Base Composition / Q000235:genetics; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000235:genetics*; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods
D009124:Muscle Proteins; D018995:Myosin Heavy Chains