Identification of Prdm genes in human corneal endothelium.
Identification of Prdm genes in human corneal endothelium.
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DOI：
10.1016/j.exer.2017.02.009
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发表时间：
2017-06
影响因子：
3.4
通讯作者：
Chittka A
中科院分区：
文献类型：
--
作者：
Rolev K;O'Donovan DG;Georgiou C;Rajan MS;Chittka A
角膜内皮细胞（corneal endothelial cells，CEC）是维持角膜基质水化和透明的重要细胞，是维持正常视力所必需的。CEC的功能障碍导致基质失代偿、透明度丧失和角膜盲。与表面上皮细胞相比，角膜内皮细胞的增殖潜力较低，导致损伤后CEC再生不良。此外，组织表现出与年龄相关的内皮细胞密度下降，细胞层重组，但没有再生。神经嵴源性CEC祖细胞增殖和分化的调控机制尚不清楚。Prdm（正调控结构域）家族的转录调控因子和染色质修饰剂对于驱动多种细胞类型的分化是重要的。许多Prdm蛋白在特定的前体细胞群体中表达，并且是其发展为完全分化表型所必需的。在目前的工作中，我们试图鉴定在人（h）CEC中特异性表达的Prdm基因家族的成员，以期开始解决它们在CEC生物学中的潜在作用，特别关注Prdm 4和5基因。通过进行半定量逆转录结合PCR扩增，我们发现除了Prdm 4和Prdm 5，Prdm 2和Prdm 10基因在hCEC中表达。我们进一步发现，培养的原代hCEC或永生化HCEC-12细胞表达CEC中发现的所有Prdm基因，但也表达额外的Prdm转录物。这种差异在从健康人角膜分离的CEC和永生化HCEC-12细胞的Prdm基因表达模式之间最为明显。我们进一步研究了Prdm 4和Prdm 5蛋白在培养的原代hCEC和HCEC-12细胞以及人尸体全角膜中的表达。Prdm 4和Prdm 5均在人角膜内皮、原代hCEC和HCEC-12细胞中表达，其特征在于Na +/K +-ATP酶的表达。我们观察到，这两种蛋白质表现出胞质（细胞内，但非核和细胞外液不同）以及核内的内皮层的定位，与Prdm 5更集中在内皮细胞的细胞核比Prdm 4。因此，我们的工作确定了新的Prdm基因特异性表达的角膜内皮细胞，这可能是重要的CEC分化和增殖的控制。对角膜内皮细胞中Prdm家族转录调控基因和染色质修饰基因进行了分析。发现Prdm 2、4、5和10基因在人CEC中表达。在CEC、离体培养的角膜内皮细胞和完整角膜中表征了Prdm 4和5蛋白表达。
Corneal endothelial cells (CECs) are essential for maintaining corneal stromal hydration and ensuring its transparency, which is necessary for normal vision. Dysfunction of CECs leads to stromal decompensation, loss of transparency and corneal blindness. Corneal endothelium has low proliferative potential compared to surface epithelial cells leading to poor regeneration of CEC following injury. Additionally, the tissue exhibits age related decline in endothelial cell density with re-organisation of the cell layer, but no regeneration. The mechanisms which control proliferation and differentiation of neural crest derived CEC progenitors are yet to be clearly elucidated. Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifiers are important for driving differentiation of a variety of cellular types. Many Prdm proteins are expressed in specific precursor cell populations and are necessary for their progression to a fully differentiated phenotype. In the present work, we sought to identify members of the Prdm gene family which are specifically expressed in human (h) CECs with a view to begin addressing their potential roles in CEC biology, focussing especially on Prdm 4 and 5 genes. By performing semi-quantitative reverse transcription coupled to PCR amplification we found that in addition to Prdm4 and Prdm5, Prdm2 and Prdm10 genes are expressed in hCECs. We further found that cultured primary hCECs or immortalised HCEC-12 cells express all of the Prdm genes found in CECs, but also express additional Prdm transcripts. This difference is most pronounced between Prdm gene expression patterns of CECs isolated from healthy human corneas and immortalised HCEC-12 cells. We further investigated Prdm 4 and Prdm 5 protein expression in cultured primary hCECs and HCEC-12 cells as well as in a human cadaveric whole cornea. Both Prdm 4 and Prdm 5 are expressed in human corneal endothelium, primary hCECs and in HCECs-12 cells, characterised by expression of the Na+/K+-ATPase. We observed that both proteins exhibit cytosolic (intracellular, but non-nuclear and distinct from extracellular fluid) as well as nuclear localisation within the endothelial layer, with Prdm 5 being more concentrated in the nuclei of the endothelial cells than Prdm 4. Thus, our work identifies novel Prdm genes specifically expressed in corneal endothelial cells which may be important in the control of CEC differentiation and proliferation. The analysis of Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifying genes in corneal endothelial cells (CECs) was undertaken. Prdm 2, 4, 5 and 10 genes were found to be expressed in human CECs. Prdm 4 and 5 protein expression was characterised in CECs, ex vivo cultured corneal endothelial cells, and the intact cornea.