The retinal pigment epithelium (RPE) is a continuous monolayer of epithelial cells located at the back of the eye that performs many functions vital for retinal preservation, including the engulfment and degradation of spent photoreceptor outer segment tips (Boulton and Pierrette Dayhaw, 2001). Photoreceptors are exposed to intense levels of light, leading to the accumulation of photo-damaged lipids and proteins (Beatty et. al., 2000). To remain functional, the outer segments must undergo a constant renewal process, in which new materials are inserted at proximal ends of outer segments, while distal photoreceptor outer segment fragments (POS) are phagocytosed by underlying RPE cells (Young, 1967). Failure of RPE phagocytosis can lead to retinal degeneration (Chaitain and Hol, 1983).There are 3 phases of RPE phagocytosis: recognition and binding of POS debris to the RPE membrane via avb5 integrin, signaling across the RPE membrane using the protein FAK, and POS engulfment using RPE F-actin (Finnemann et al., 1997; Law et al., 2007).
The objective of this study was to characterize cortactin expression in RPE cells. The CTTN gene consists of 18 exons, which code for the protein cortactin. (Kirkbride et al., 2011). Cortactin regulates branched F-actin assembly by interacting with the Arp2/3 complex at its N-terminus, binding to F-actin at its cortactin repeats, and binding N-WASP at its C-terminal SH3 domain (Kirkbride et al., 2011). The aim of this study was to characterize cortactin expression in RPE cells since this protein is important for F-actin assembly, as well as in the neural retina to determine if cortactin’s expression is unique to the RPE. (Lua and Low, 2004). In addition, cortactin has been reported to interact with FAK, a key component of the RPE phagocytic pathway. (Tomar et. al., 2012) Since FAK is activated by the POS recognition receptor avb5 integrin, the goal of this study was also to determine if cortactin expression is impacted by the absence of the avb5 integrin.
Materials and Methods
3 month-old Wild-type, β5 integrin knockout, and JNK2 knockout mice were sacrificed by CO2 asphyxiation at the same hour of the day. Eyeballs were carefully enucleated and rinsed in HBSS without Ca2+ and Mg2+. The lens and vitreous humor were dissected out from each eye. Retinas were then removed from each eye, leaving a remaining eyecup containing the RPE/choroid layers. For one sample, three retinas or three eyecups were pooled together in RLT lysis buffer plus β-mercaptoethanol (Quiagen kit) and frozen at -80° C until further RNA extraction.
All lysate samples were purified from genomic DNA by a Qiashredder column from Qiagen. RNA was then extracted following the Qiagen RNeasy plus mini kit, following the manufacturer protocol. The Purity and concentration of each sample were then analyzed by spectrophotometry, and a 5ng/ul solution stock were made and stored at -20° C.
RT-PCR was performed using the Qiagen One-Step RT-PCR kit. Ten nanograms of RNA were amplified in a 10ul reaction. RT PCR for cortactin in eyecups and neural retinas was performed with primers spanning exons 2-6, 3-11, and 10-18. An RT PCR for the housekeeping gene GAPDH was performed with 0.5uM of primers. Products were analyzed in a 1% agarose gel with Ethidium bromide. Products were then gel purified using the QIAquick gel extraction kit and set out for Sanger sequencing by Genewiz.
To determine if cortactin expression is impacted by the absence of the avb5 integrin, an RT PCR for cortactin was performed in wild-type and b5 integrin knockout mice eyecups and neural retinas using primer pairs that spanned exons 2-6, 3-11, and 10-18. The results indicate that the level of cortactin transcript appears to be higher in β5 integrin knockout eyecups in comparison to wild type eyecups (Figure 1). An increase in cortactin expression in β5 integrin knockout eyecups was not observed in the region amplified using primer pairs for exons 2-6. This increase was not observed in the neural retinas. Moreover, the results in Figure 1 indicated the presence of two bands in the region amplified using primer pairs spanning exons 10-18.
In order to determine whether two alternative cortactin transcripts were being produced, another RT PCR for cortactin was performed using primer pairs spanning exons 10-18. This analysis utilized eyecups and neural retinas from wild-type, β5 integrin knockout mice as well as JNK2 knockout mice, which were originally used to test primer pairs, but were employed here as controls to determine whether the increase in cortactin expression observed in β5 integrin knockout eyecups was specific to that knockout. Two bands were once again produced in both eyecups and neural retinas of wild-type, β5 integrin knockout, and JNK2 knockout mice (Figure 2). Sequencing analysis revealed two cortactin alternative transcripts that differed by 111 base pairs, corresponding to exon 11 of cortactin. In addition, the results indicated higher cortactin expression in β5 integrin knockout eyecups in comparison to wild type eyecups, or JNK2 knockout eyecups (Figure 2). An RT-PCR for the housekeeping GAPDH gene confirmed the increase in cortactin expression.
The purpose of this study was to characterize cortactin expression in RPE cells as well as to determine if cortactin expression is impacted by the absence of the avb5 integrin. The results of this study d indicate that the expression of cortactin appears to be higher in β5 integrin knockout eyecups in comparison to wild type eyecups. An increase in cortactin expression in β5 integrin knockout eyecups was not observed in the region amplified using primer pairs for exons 2-6. One explanation for such an observation could be that a cortactin transcript may be produced beginning at exon 3 and may be transcribed more frequently than the transcript produced beginning at exon 2. Moreover, the increase in cortactin expression observed in eyecups was not observed in the neural retinas, suggesting that this increase in cortactin expression in β5 integrin knockout mice was specific to tissues of the eyecup where the RPE cells reside.
Moreover, this study identified the production of two cortactin transcript variants in eyecups and neural retinas of all mice examined, one that is missing exon 11 (111bp) and one that contains exon 11. Exon 11 encodes a cortactin repeat of 37 amino acids in length that is responsible for binding F-actin (Van Rossum et al., 2003) (Figure 3). Future studies could examine the protein expression of these alternative transcripts in both the retina and RPE as well as their physiological roles in these tissue types.
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