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Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells
Published on: December 12, 2014
Gene expression profile changes caused by the dysfunction of Mer during retinal pigment epithelium phagocytosis
Yan-yun Chen1, Qing-jun Lu, Qing-xian Lu
1Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
Background:
Studies indicated that Mer might be the main contributor to the specific internalization of photoreceptor outer segments (POS) in retinal pigment epithelium (RPE). It is very important to understand the mechanism of POS phagocytosis under the pathway of Mer and its ligands. The objective of this study was to identify changes in gene expression profiles caused by Mer gene knockout (Mer-/-) during phagocytosis of POS in RPE.
Methods:
RPE from both Mer-/- and wild-type (WT) mice were isolated and cultured to the 3rd passage. POS were subjected to culture medium with 20 nmol/L Gas6 and protein S to activate specific mer-mediated phagocytosis. RPE phagocytosis was evaluated by phagocytosis assays and differential gene expression identified by microarray at 3 and 12 hours; the 0-hour time point served as the control. Three independent samples for each Mer-/- or WT RPE were subjected to the same protocol of microarray. Five genes were confirmed by real-time quantitative PCR (QPCR).
Results:
The Mer-/- RPE had less internalized POS than WT RPE after both 3 and 12 hours in phagocytosis assay. Compared to WT RPE and the 0-hour control, 38 and 45 different known genes were increased and 68 and 59 known genes were decreased in Mer-/- RPE after 3 and 12 hours, respectively. Abnormal POS phagocytosis in Mer-/- RPE was associated with significant gene expression changes in, for example, signal transduction (WNT, MAPK), phagocytosis (Vav3, Hsd11b1), cytoskeleton components (Myo7a), and metabolism, in a time-specific manner. QPCR results showed Vav3, Hsd11b1, Myo7a, Rtn2 and Itga8 in those independent samples were consistent with microarray.
Conclusion:
Gene expression profiles modulated in a time-specific manner in Mer-/- RPE indicate a possible internalization mechanism for abnormal POS phagocytosis, which gives insight into the mechanism of retinitis pigmentosa caused by the mutation of MerTK in humans.
Insights
Mer knockout in retinal pigment epithelium (RPE) impairs photoreceptor outer segment (POS) clearance, revealing gene expression changes linked to abnormal phagocytosis and retinitis pigmentosa.
Area of Science:
- Ophthalmology
- Molecular Biology
- Cell Biology
Background:
- Mer receptor tyrosine kinase is crucial for photoreceptor outer segment (POS) clearance by retinal pigment epithelium (RPE).
- Understanding Mer-mediated POS phagocytosis is vital for retinal health.
Purpose of the Study:
- To investigate gene expression changes in RPE lacking the Mer gene (Mer-/-) during POS phagocytosis.
- To elucidate the role of Mer in POS internalization.
Main Methods:
- Primary RPE from Mer-/- and wild-type (WT) mice were cultured.
- POS were incubated with RPE in the presence of Mer agonists (Gas6 and protein S).
- Phagocytosis assays and microarray analysis were performed at 3 and 12 hours.
Main Results:
- Mer-/- RPE exhibited significantly reduced POS internalization compared to WT RPE.
- Microarray analysis revealed differential gene expression in Mer-/- RPE, affecting pathways like signal transduction, phagocytosis, and metabolism.
- Key genes identified include Vav3, Hsd11b1, and Myo7a, with findings confirmed by quantitative PCR.
Conclusions:
- Altered gene expression profiles in Mer-/- RPE suggest a mechanism for impaired POS phagocytosis.
- These findings provide insights into MerTK mutations causing retinitis pigmentosa in humans.
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