Lens α-, β-, γ-crystallins Expression In Mammalian Corneas

PURPOSE. How corneal transparency is formed/maintained remains largely unclear. A group of enzymes which are referred to as enzymatic or corneal crystallins were proposed to contribute to corneal transparency in invertebrates and vertebrates. This study investigated whether the three classical lens crystallins, namelyα-,β- andγ-crystallins, play any role in mouse and human corneas.METHODS. Two inflammation corneal neovascularization (CNV) models, namely suture-induced CNV (S-CNV) and chemical burn-induced CNV (CB-CNV) were set up in Balb/c and C57Bl/6 mice. The expression of lens crystallins and corneal crystallins in corneas or in cultured corneal cells were detected at mRNA level by whole-genome microarray profiling or reverse transcription quantitative real-time PCR (RT-Q-PCR), as well as at protein level by immunohistochemistry (IHC) or western blotting.RESULTS. Many lens crystallin genes transcripts were present in corneas or cultured corneal cells abundantly that could be detected by microarray profiling or RT-Q-PCR, and expression of them at protein level in denovo cornea or cultured corneal cells were confirmed with IHC or western blotting of several representatives likeαA-,β- andγ-crystallins. In the S-CNV and CB-CNV models, lens crystallin genes showed time-course dependent and strain dependent changes in a pattern that differed from corneal crystallin genes. Lens crystallin gene expression in cultured corneal cells responded to exogenous oxidative or inflammatory stimuli like H2O2 or lipopolysaccharide. CONCLUSION. Lens crystallins are involved in the maintenance and loss of corneal transparency in mammals, and further studies of related mechanisms may help unravel the mystery of corneal transparency. PURPOSE: To evaluate the suitability of common housekeeping genes (HKGs) for use in reverse transcription quantitative real-time PCR (RT-Q-PCR) assays of the cornea in various murine disease models.METHODS: Corneal disease models studied were: 1) corneal neovascularization (CorNV) induced by suture or chemical burn, 2) corneal infection with Candida albicans or Aspergillus fumigatus by intrastromal injection of live spores, and 3) perforating corneal injury (PCI) in Balb/c mice or C57BL/6 mice. Expression of 8 HKGs (viz. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), lactate dehydrogenase A (LDHA), ribosomal protein L5 (RPL5), ubiquitin C (UBC), peptidylprolyl isomerase A (PPIA), TATA-box binding protein, hypoxanthine guanine phosphoribosyl transferase (HPRT1)) in the cornea were measured at various time points by microarray hybridisation or RT-Q-PCR and the data analyzed using geNorm and NormFinder.RESULTS: Microarray results showed that under the CorNV condition the expression stability ofthe 8 HKGs decreased in order of PPIA>RPL5>HPRT1>ACTB>UBC>TBP1>GAPDH>LDHA. RT-Q-PCR analyses demonstrated that expression of none of the 8 HKGs remained stable under all conditions, while GAPDH and ACTB were among the least stably expressed markers under most conditions. Both geNorm and NormFinder analyses proposed best HKGs or HKG combinations that differ between the various models. NormFinder proposed PPIA as best HKG for three CorNV models and PCI model, as well as UBC for two fungal keratitis models. geNorm analysis demonstrated that a similar model in different mice strains or caused by different stimuli may require different HKGs or HKG pairs for the best normalization. Namely, geNorm proposed PPIA&HRPT1 and PPIA&RPL5 pairs for chemical burn-induced CorNV in Balb/c and C57BL/6 mice respectively, while UBC&HPRT1 and UBC&LDHA were best for Candida and Aspergillus induced keratitis in Balb/c mice respectively. CONCLUSION: When RT-Q-PCR is designed for studies of gene expression in murine cornea, preselection of situation-specific reference genes is recommended. In the absence of knowledge about situation-specific HKGs, PPIA and UBC, either alone or in combination with HPRT1 or RPL5, can be employed.