title: Identification of Prdm genes in human corneal endothelium.
abstract: 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.
authors.affiliation: Anglia Ruskin University, Department of Biomedical and Forensic Sciences and the Vision & Eye Research Unit, Cambridge CB1 1PT, United Kingdom. Electronic address: Kostadin.Rolev@anglia.ac.uk.
authors.forename: Kostadin
authors.initials: K
authors.lastname: Rolev
authors.affiliation: Dept. of Histopathology, Cambridge University Hospitals, Hills Road, Cambridge, Cambridgeshire CB2 0QQ, United Kingdom. Electronic address: dominic.odonovan@addenbrookes.nhs.uk.
authors.forename: Dominic G
authors.initials: DG
authors.lastname: O'Donovan
authors.affiliation: The Wolfson Institute for Biomedical Research, Division of Medicine, UCL, Gower St, London WC1E 6BT, United Kingdom. Electronic address: c.georgiou@qmul.ac.uk.
authors.forename: Christiana
authors.initials: C
authors.lastname: Georgiou
authors.affiliation: Anglia Ruskin University, Department of Biomedical and Forensic Sciences and the Vision & Eye Research Unit, Cambridge CB1 1PT, United Kingdom; Department of Ophthalmology, Cambridge University Hospitals, Hills Road, Cambridge, Cambridgeshire CB2 0QQ, United Kingdom. Electronic address: madhavan.rajan@addenbrookes.nhs.uk.
authors.forename: Madhavan S
authors.initials: MS
authors.lastname: Rajan
authors.affiliation: The Wolfson Institute for Biomedical Research, Division of Medicine, UCL, Gower St, London WC1E 6BT, United Kingdom. Electronic address: a.chittka@qmul.ac.uk.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Chittka
mesh_terms: D002454:Cell Differentiation; D002478:Cells, Cultured; D003316:Corneal Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004728:Endothelium, Corneal / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D012313:RNA / Q000235:genetics*; D012038:Regeneration / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C403744:PRDM4 protein, human; C487762:PRDM5 protein, human; D014157:Transcription Factors; D012313:RNA
keywords: Cornea; Corneal endothelial cells; Prdm (Prdm refers to the protein, Prdm refers to mRNA)