title: Phenotypic switching induced by damaged matrix is associated with DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth muscle cells (SMC).
abstract: Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0-2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O2 (balanced 5% CO2 and 95% N2) over 48 hours. Inhibitors were applied 2-3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/- hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.
authors.affiliation: Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada ; Division of Urology, Hospital for Sick Children, Toronto, Ontario, Canada ; Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jia-Xin
authors.initials: JX
authors.lastname: Jiang
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Aitken
authors.forename: Chris
authors.initials: C
authors.lastname: Sotiropoulos
authors.forename: Chris
authors.initials: C
authors.lastname: Sotiropolous
authors.forename: Tyler
authors.initials: T
authors.lastname: Kirwan
authors.forename: Trupti
authors.initials: T
authors.lastname: Panchal
authors.forename: Nicole
authors.initials: N
authors.lastname: Zhang
authors.forename: Shuye
authors.initials: S
authors.lastname: Pu
authors.forename: Shoshana
authors.initials: S
authors.lastname: Wodak
authors.forename: Cornelia
authors.initials: C
authors.lastname: Tolg
authors.forename: Darius J
authors.initials: DJ
authors.lastname: Bägli
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D002452:Cell Count; D054337:Cell Dedifferentiation / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000737:chemistry / Q000494:pharmacology; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects; D000090205:DNA Methyltransferase 3A; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D010641:Phenotype; D014176:Protein Biosynthesis / Q000187:drug effects; D011489:Protein Denaturation; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: C000717967:DNMT3A protein, human; D003094:Collagen; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A