Altered sensitivity to single-strand-specific reagents associated with the genomic vascular smooth muscle alpha-actin promoter during myofibroblast differentiation.
Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor beta1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle alpha-actin mRNA and protein. Because transcriptional regulation of the smooth muscle alpha-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Puralpha, Purbeta, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor beta1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities. These results suggest that activation of the endogenous smooth muscle alpha-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding.
Becker
N A
NA
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Kelm
R J
RJ
Vrana
J A
JA
Getz
M J
MJ
Maher
L J
LJ
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D000077214:Becaplermin; D002454:Cell Differentiation / Q000187:drug effects; D004277:DNA, Single-Stranded / Q000187:drug effects*; D004742:Enhancer Elements, Genetic; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D020574:Proto-Oncogene Proteins c-sis; D016385:TATA Box; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D000199:Actins; D004277:DNA, Single-Stranded; D007328:Insulin; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D016212:Transforming Growth Factor beta; D000077214:Becaplermin; D004815:Epidermal Growth Factor