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Modulation of DNA methylation and phenotypic switching in Smooth Muscle Cells by the extracellular matrix microenvironment

机译：细胞外基质微环境对平滑肌细胞DNA甲基化和表型转换的调节

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BackgroundPartial bladder outlet obstruction due to neurogenic bladderor mechanical obstruction is common amongst thepopulation and can cause bladder injury and dysfunction.Bladder Smooth Muscle Cells (BSMCs) undergo phenotypicchanges such as hyper-proliferation, de-differentiationand altered expression of integrins and ECM proteins.[1]Extracellular matrix changes are often crucial incitingevents for fibroproliferative disease.[2] Epigenetic change,specifically DNA methylation, may be important factorsunderlying the persistent fibroproliferative phenotype. Previously,damaged matrix (heat-denatured collagen, DNC)induced hyper-proliferation of bladder smooth musclecells (BSMC) and the phenotype was not reverted upon areturn to normal matrix. [3]We examined the dependencyof matrix-induced fibroproliferation and SMC phenotypeon DNA methyltransferase activity. The cooperativity ofmatrix with other inciting stimuli (growth factors, hypoxiaand strain) associated with bladder obstruction was alsoexamined.Material and methodsPrimary cultures of neonatal rat BSMC (early passage of0-2) and human BSMC were plated on 12-, 24-well or10cm culture plates that are pre-coated with either type Ibovine native collagen (NC) or DNC (heat-denatured NC).Hypoxia was induced at 3% O2 and 5%CO2 with a balanceof N2. Mechanical strain was applied with slow rampingup to a final 5% elongation over 16 hours. Inhibitors wereadded 2-3 hours after plating and remained for 48hrs.Cells were fixed, stained for DNA methyltransferase 3A(DNMT3A), a-Smooth muscle actin (a-SMA) and otherSMC differentiation markers. Intensities and cell numberswere analysed on ImageJ Finally, Illumina 450K array ofCpG sites was performed on bisulfite converted DNAfrom human smooth muscle cells on DNC vs. NC.ResultsDNC exposure significantly increased the translocalizationof DNMT3A into the nucleus of BSMC, in contrast to NCcells, which expressed only cytoplasmic DNMT3A. Theincrease in nuclear expression of DNMT3A was coupledwith decreased expression level of a-SMA. Hypoxia withDNC increased DNMT3A nuclear expression, but mechanicalstrain only mildly increased DNMT3A expression. OnDNC cultures, AG490 (JAK2/STAT inhibitor) significantlyreduced DNMT3A nuclear localization (P=0.001), withoutchanging a-SMA expression and proliferation. Aza-cytidinesuppressed hyper-proliferation of BSMCs cultured on DNCwithout affecting basal proliferation on NC. On damagedmatrix, Sonic hedgehog (SHH) upregulated expression ofa-SMA, but did not alter proliferation. However, SHHincreased absolute levels of both nuclear and cytoplasmicDNMT3A. Followed by rigorous multiple testing, we discoveredsignificant changes in methylation status for7 genes in the Illumina 450K array.ConclusionsMatrix exquisitely regulates DNMT3A localization andexpression, and influences differentiation in BSMCsexposed to denatured matrix +/- growth factors or SHH.That nuclear expression of DNMT does not always correspondto decreased a-SMA expression suggests thatDNA methylation may not directly act as a dedifferentiatingfactor, as it appears to influence both proliferation/loss of differentiation on DNC as well as differentiationby SHH. Future work will examine how expression of other SMC markers is affected by shRNA knockdown ofDNMTs.
机译：背景由于神经源性膀胱引起的部分膀胱出口梗阻或机械性阻塞在人群中很常见，并可能导致膀胱损伤和功能障碍。膀胱平滑肌细胞（BSMC）发生表型改变，例如过度增殖，去分化以及整合素和ECM蛋白的表达改变。[1] ]细胞外基质变化通常是引起纤维增生性疾病的关键诱因。[2]表观遗传的改变，特别是DNA甲基化，可能是持续的纤维增生表型的重要因素。以前，受损的基质（热变性胶原蛋白，DNC）引起膀胱平滑肌细胞（BSMC）过度增殖，表型在恢复为正常基质后不会恢复。 [3]我们检查了基质诱导的纤维增生和SMC表型对DNA甲基转移酶活性的依赖性。还检查了基质与其他与膀胱阻塞相关的刺激性刺激（生长因子，低氧和劳损）的协同作用。材料和方法将新生大鼠BSMC（0-2早期通过）和人BSMC的原代培养物分别铺在12、24孔或10cm培养上预先用Ibovine天然胶原（NC）或DNC（热变性NC）涂层的板。缺氧在3％O2和5％CO2且其余为N2的条件下引起。在16小时内以缓慢的斜度施加机械应变，直至最终5％的伸长率。铺板后2-3小时加入抑制剂，并保持48小时。固定细胞，对DNA甲基转移酶3A（DNMT3A），α-平滑肌肌动蛋白（a-SMA）和其他SMC分化标记物染色。在ImageJ上分析了强度和细胞数量。最后，在DNC与NC上，对来自人平滑肌细胞的亚硫酸氢盐转化的DNA进行了Illumina 450K CpG位点阵列的分析。仅细胞质的DNMT3A。 DNMT3A核表达增加与α-SMA表达水平降低有关。缺氧与DNC增加DNMT3A核表达，但机械应变仅轻度增加DNMT3A表达。在OnDNC培养物中，AG490（JAK2 / STAT抑制剂）显着降低了DNMT3A核定位（P = 0.001），而没有改变a-SMA的表达和增殖。氮杂胞苷抑制了在DNC上培养的BSMC的过度增殖，而不会影响NC的基础增殖。在受损的基质上，声波刺猬（SHH）上调了a-SMA的表达，但没有改变增殖。但是，SHH增加了核和细胞质DNMT3A的绝对水平。经过严格的多重测试，我们发现Illumina 450K阵列中7个基因的甲基化状态发生了显着变化。结论基质可以很好地调节DNMT3A的定位和表达，并影响暴露于变性基质+/-生长因子或SHH的BSMC的分化.DNMT的核表达没有总是与降低的a-SMA表达相对应，表明DNA甲基化可能不直接充当去分化因子，因为它似乎影响DNC分化的增殖/丧失以及SHH的分化。未来的工作将研究DNMT的shRNA敲除如何影响其他SMC标记的表达。

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