Department of Cellular and Molecular Medicine, The University of Arizona, Tucson, AZ 85724, USA.
Dis Model Mech. 2013 May;6(3):632-42. doi: 10.1242/dmm.010595. Epub 2012 Dec 20.
Endothelia in the atrioventricular (AV) canal of the developing heart undergo a prototypical epithelial mesenchymal transition (EMT) to begin heart valve formation. Using an in vitro invasion assay, an extracellular matrix protein, Olfactomedin-1 (OLFM1), was found to increase mesenchymal cell numbers in AV canals from embryonic chick hearts. Treatment with both anti-OLFM1 antibody and siRNA targeting OLFM1 inhibits mesenchymal cell formation. OLFM1 does not alter cell proliferation, migration or apoptosis. Dispersion, but lack of invasion in the presence of inhibiting antibody, identifies a specific role for OLFM1 in cell invasion during EMT. This role is conserved in other epithelia, as OLFM1 similarly enhances invasion by MDCK epithelial cells in a transwell assay. Synergy is observed when TGFβ2 and OLFM1 are added to MDCK cell cultures, indicating that OLFM-1 activity is cooperative with TGFβ. Inhibition of both OLFM1 and TGFβ in heart invasion assays shows a similar cooperative role during development. To explore OLFM1 activity during EMT, representative EMT markers were examined. Effects of OLFM1 protein and anti-OLFM1 on transcripts of cell-cell adhesion molecules and the transcription factors Snail-1, Snail-2, Twist1 and Sox-9 argue that OLFM1 does not initiate EMT. Rather, regulation of transcripts of Zeb1 and Zeb2, secreted proteases and mesenchymal cell markers by both OLFM1 and anti-OLFM1 is consistent with regulation of the cell invasion step of EMT. We conclude that OLFM1 is present and necessary during EMT in the embryonic chick heart. Its role in cell invasion and mesenchymal cell gene regulation suggests an invasion checkpoint in EMT where OLFM1 acts to promote cell invasion into the three-dimensional matrix.
心房间隔（AV）的内皮细胞经历典型的上皮间质转化（EMT），开始心脏瓣膜的形成。利用体外侵袭实验，发现一种细胞外基质蛋白 Olfactomedin-1（OLFM1）可增加胚胎鸡心 AV 管中的间质细胞数量。用抗 OLFM1 抗体和针对 OLFM1 的 siRNA 处理均能抑制间质细胞的形成。OLFM1 不改变细胞增殖、迁移或凋亡。在存在抑制抗体的情况下，细胞的分散而非侵袭，表明 OLFM1 在 EMT 中的细胞侵袭中具有特定作用。这种作用在其他上皮细胞中是保守的，因为 OLFM1 同样增强了 MDCK 上皮细胞在 Transwell 测定中的侵袭能力。当 TGFβ2 和 OLFM1 添加到 MDCK 细胞培养物中时，观察到协同作用，表明 OLFM-1 活性与 TGFβ 协同作用。在心脏侵袭实验中，抑制 OLFM1 和 TGFβ 显示出在发育过程中类似的协同作用。为了探索 EMT 期间 OLFM1 的活性，检查了 EMT 的代表性标志物。OLFM1 蛋白和抗 OLFM1 对细胞-细胞黏附分子的转录物和转录因子 Snail-1、Snail-2、Twist1 和 Sox-9 的影响表明，OLFM1 不会引发 EMT。相反，OLFM1 和抗 OLFM1 对 Zeb1 和 Zeb2 的转录物的调节，以及分泌的蛋白酶和间充质细胞标志物，与 EMT 中细胞侵袭步骤的调节一致。我们得出结论，OLFM1 在胚胎鸡心中 EMT 期间存在且是必需的。它在细胞侵袭和间质细胞基因调控中的作用表明 EMT 中存在侵袭检查点，OLFM1 在此处发挥作用，促进细胞侵袭到三维基质中。
