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. 2008 Nov 15;22(22):3147-57.
doi: 10.1101/gad.1700108. Epub 2008 Nov 7.

Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium

Jin Woo Kim  1 , Kyung Hwa Kang, Patrick Burrola, Tak W Mak, Greg Lemke
Affiliations

Retinal degeneration triggered by inactivation of PTEN in the retinal pigment epithelium

Jin Woo Kim et al. Genes Dev. .

Abstract

Adhesion between epithelial cells mediates apical-basal polarization, cell proliferation, and survival, and defects in adhesion junctions are associated with abnormalities from degeneration to cancer. We found that the maintenance of specialized adhesions between cells of the retinal pigment epithelium (RPE) requires the phosphatase PTEN. RPE-specific deletion of the mouse pten gene results in RPE cells that fail to maintain basolateral adhesions, undergo an epithelial-to-mesenchymal transition (EMT), and subsequently migrate out of the retina entirely. These events in turn lead to the progressive death of photoreceptors. The C-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding domain of PTEN is essential for the maintenance of RPE cell junctional integrity. Inactivation of PTEN, and loss of its interaction with junctional proteins, are also evident in RPE cells isolated from ccr2(-/-) mice and from mice subjected to oxidative damage, both of which display age-related macular degeneration (AMD). Together, these results highlight an essential role for PTEN in normal RPE cell function and in the response of these cells to oxidative stress.

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Figures

Figure 1.
Figure 1.
RPE-specific deletion of mouse PTEN. (A) Expression of PTEN in the adult retina. A section of a 3-mo-old mouse eye was stained with mouse anti-PTEN antibody (green) with counter-nuclear staining with DAPI (blue, left panel). PTEN is enriched in the RPE layer where the signal for anti-phospho-Akt(S473) (pAkt; red, middle and right panels) is lowest. Boxed areas in the middle panel are enlarged in right panels to provide closer views to the costaining images of PTEN and pAkt at RPE–photoreceptor boundary (top right panel) and at GCL (bottom right panel). Upper and lower dashed lines represent basal and apical extents of the RPE layer, respectively. (OS) Photoreceptor outer segment; (IS) inner segment; (ONL) outer nuclear layer (photoreceptor nuclei); (OPL) outer plexiform layer; (INL) inner nuclear layer; (IPL) inner plexiform layer; (GCL) retinal ganglion cell layer. (B) PTEN localizes to apical microvilli (*) and basolateral areas (arrowhead) of RPE cells in P8 fl/fl mice. This signal is absent in fl/fl;T1Cre RPE cells, while PTEN in the choroids, sclera, or retina is intact. Upper and lower dashed lines represent basal and apical extents of the RPE layer, respectively. (C, top panel) RPE cells were isolated from P8 eyes, and Cre-mediated deletion of the genomic fragment spanning exons 4 and 5 of PTEN gene (Δ4–5) (Suzuki et al. 2001) was examined by PCR. The middle panel displays corresponding genotyping PCR using tail DNA, where Cre is not expressed. (Bottom panel) Loss of PTEN in the RPE was confirmed by immunoblot (IB) with PTEN antibody. Bars, 50 μm.
Figure 2.
Figure 2.
Progressive RPE loss in fl/fl;T1Cre eyes. (A–F) H&E staining of fl/fl and fl/fl;T1Cre eye sections at the indicated postnatal (P) days. Images in E′ and F′ are lateral views of 3-mo-old f/fl and fl/fl;T1Cre eyes, respectively. (G, H) Osmium tetroxide staining of 1-mo-old f/fl and fl/fl;T1Cre eye sections. (I) Total RPE cells were purified from eyes at the indicated postnatal day, dissociated, and counted. (n ≥ 6) (see the Materials and Methods for details). Bars: A–F, 50 μm; G,H, 10 μm.
Figure 3.
Figure 3.
Impaired intercellular adhesion between PTEN-deficient RPE cells. EM images from P28 fl/+;T1Cre (A,D), and fl/fl;T1Cre (B,C,E,F) mouse eyes. fl/+;T1Cre RPE cells form well-organized basolateral appositions (bracket in A), but PTEN-deficient RPE lose these appositions, resulting in large intercellular vacuoles (bracket in B). Basal folds of the mutant RPE cells, which contact Bruch’s membrane (BM), degenerate (B), and many RPE cells contain enlarged, dysmorphic pigment vesicles (arrowheads in C). (D,E) Magnified EM images of A, B, and E are shown in D, E, and F, respectively. (TJ) Tight junction; (MV) microvilli; (OS) outer segments; (CRD) choroid. Bars, 1 μm.
Figure 4.
Figure 4.
The PDZ-binding domain of PTEN is essential for RPE AJs. RPE cells isolated from fl/fl;T1Cre mice were cultured on the Matrigel-coated transwell plates, and either mock-infected (A) or infected with retroviruses expressing wild-type PTEN (B), a lipid phosphatase-defective PTEN(G129E) mutant (C), a phosphatase-null PTEN(G129R) mutant (D), a mutant lacking lipid-binding C2 domains (M-CBR3; E), or a truncated PTEN mutant lacking the C-terminal PDZ-binding domain (Q399X; F). The localization of β-catenin in RPE cells was examined at 36 h of post-infection by immunostaining with anti-β-catenin antibody (red) and counterstaining with anti-PTEN antibody (green). For each panel, the fine dashed line indicates the position of the section for the orthogonal Z-stack displayed at the bottom of the panel, and arrowheads on the dashed line correspond to arrowheads in the bottom panel. Api and bas indicate apical and basal surfaces of the Z-stack. Bar, 50 μm.
Figure 5.
Figure 5.
PTEN perturbation in RPE cells undergoing chemically and genetically induced retinal degeneration. (A) Frozen sections (10 μm) of 11-mo-old wild-type or ccr2−/− mouse eyes were stained with antibodies against PTEN, β-catenin, or Ezrin. (B) Relative proteins levels in RPE cells isolated from 11- to 12-mo-old wild-type or ccr2−/− mice were accessed by Western blotting with anti-phospho-PTEN(S380/T382/T383) (pPTEN), anti-PTEN (PTEN), anti-phospho-Akt(S473) (pAkt), anti-Akt (Akt), α-crystallin, or β-actin antibody. (C) C57/BL6 wild-type mice (3 mo old) were intravenously injected with NaIO3 (30 mg/kg), and the levels of pPTEN, pAkt, and β-catenin in RPE cells were monitored by immunostaining after 24 h. (D) The levels of pPTEN, PTEN, pAkt, Akt, α-crystallin, or β-actin in RPE cells from the NaIO3-injected mice were assessed by Western blotting. Arrowheads in A and C indicate the area magnified in each inset. Bar, 50 μm.
Figure 6.
Figure 6.
PTEN-deficient RPE cells lose epithelial properties and undergo an EMT. (A) Comparative analyses of gene expression patterns between wild-type (WT) and PTEN-deficient (KO) RPE cells using Q-PCR and microarrays (see Supplemental Table S1). Results are average values from triplicate (Q-PCR) or duplicate (microarray) analyses. (B) Equivalent amounts of total cell lysates from wild-type (fl/fl) or PTEN-deficient (fl/fl;T1Cre) mouse RPE cells were also analyzed by Western blotting. (C) RPE cells isolated from fl/fl, fl/+;T1Cre, or fl/fl;T1Cre mice were grown on Matrigel transwell plates for 2 wk, and the number of cells migrating into and through the Matrigel membrane were counted (see the Materials and Methods). (D) RPE cells from fl/fl mice form an epithelial sheet on Matrigel matrix, with an intact microfilament network, as monitored by FITC-labeled phalloidin (green), at cell-to-cell adhesion sites. Stress fibers were more prominent in PTEN-deficient RPE cells isolated from fl/fl;T1Cre mice. Nuclei of RPE cells in 3D culture are shown by DAPI staining (blue). Bar, 50 μm. (E-cad) E-cadherin; (β-catn) β-catenin; (DSP) desmoplakin; (Crb3) Crumbs 3; (α-SMA) α-smooth muscle actin; (vmntn) vimentin.
Figure 7.
Figure 7.
Schematic diagram of pathological signaling pathways in PTEN-inactivated RPE cells. (Top) PTEN normally functions as a component of AJs in healthy RPE cells, through its interaction with junctional PDZ proteins. This requires the C-terminal PDZ-binding domain of PTEN. (Bottom) PTEN is lost from junctional structures by phosphorylational inactivation upon oxidative stress or somatic mutation. β-Catenin is then released from dissociated junctional complexes, and enters the nucleus. RPE cells are thereby transformed into highly migratory mesenchymal cells, through β-catenin/TCF induction of new mesenchymal genes.
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References

    1. Aberle H., Bauer A., Stappert J., Kispert A., Kemler R. β-Catenin is a target for the ubiquitin–proteasome pathway. EMBO J. 1997;16:3797–3804. - PMC - PubMed
    1. Adey N.B., Huang L., Ormonde P.A., Baumgard M.L., Pero R., Byreddy D.V., Tavtigian S.V., Bartel P.L. Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding. Cancer Res. 2000;60:35–37. - PubMed
    1. Adler R., Curcio C., Hicks D., Price D., Wong F. Cell death in age-related macular degeneration. Mol. Vis. 1999;5:31. - PubMed
    1. Ambati J., Anand A., Fernandez S., Sakurai E., Lynn B.C., Kuziel W.A., Rollins B.J., Ambati B.K. An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat. Med. 2003;9:1390–1397. - PubMed
    1. Bailey T.A., Kanuga N., Romero I.A., Greenwood J., Luthert P.J., Cheetham M.E. Oxidative stress affects the junctional integrity of retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2004;45:675–684. - PubMed

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