TY - JOUR
T1 - Methylation of the estrogen receptor-α gene promoter is selectively increased in proliferating human aortic smooth muscle cells
AU - Ying, Anita K.
AU - Hassanain, Hamdy H.
AU - Roos, Christine M.
AU - Smiraglia, Dominic J.
AU - Issa, Jean Pierre J.
AU - Michler, Robert E.
AU - Caligiuri, Michael
AU - Plass, Christoph
AU - Goldschmidt-Clermont, Pascal J.
N1 - Funding Information:
We thank Julie Popovich and Julia Eisel for technical assistance. AKY was supported in part by the Stanley J. Sarnoff Endowment for Cardiovascular Science, CP by NIH grant GM58269, and DJS by NCI T32 CA09338. Support for this work was also provided by the Scleroderma Research Foundation and by NIH grant P30 CA16058.
PY - 2000/4
Y1 - 2000/4
N2 - Objective: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-α gene (ERα) promoter blocks the expression of ERα, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERα is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. Methods: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERα in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). Results: We provide evidence that methylation of the ERα in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERα promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS- M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERα and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. Conclusions: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERα, could contribute to the switch in phenotype observed in these cells. (C) 2000 Elsevier Science B.V.
AB - Objective: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-α gene (ERα) promoter blocks the expression of ERα, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERα is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. Methods: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERα in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). Results: We provide evidence that methylation of the ERα in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERα promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS- M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERα and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. Conclusions: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERα, could contribute to the switch in phenotype observed in these cells. (C) 2000 Elsevier Science B.V.
KW - Artherosclerosis
KW - Receptors
KW - Smooth muscle
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U2 - 10.1016/S0008-6363(00)00004-3
DO - 10.1016/S0008-6363(00)00004-3
M3 - Article
C2 - 10727665
AN - SCOPUS:0034060168
SN - 0008-6363
VL - 46
SP - 172
EP - 179
JO - Cardiovascular Research
JF - Cardiovascular Research
IS - 1
ER -