Naitoh H et al. (2017), Upregulation of matrix metalloproteinase trigge...

XB-ART-53175

Dev Neurobiol 2017 Sep 01;779:1086-1100. doi: 10.1002/dneu.22497.

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Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration.

Naitoh H , Suganuma Y , Ueda Y , Sato T , Hiramuki Y , Fujisawa-Sehara A , Taketani S , Araki M .

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In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10-PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL-1β and TNF-α, were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086-1100, 2017.

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Species referenced: Xenopus laevis
Genes referenced: cacna1b mmp1 mmp7 mmp8 pax6 rax rpe tnf

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	Figure 1 qPCR analysis of expression of Xenopus Mmp7, Mmp8, Mmp9, Mmp18 in the RPE1choroid tissues. Tissues containing RPE and choroid were isolated from Xenopus eyes on Days 1, 5, and 10 after retinal removal, and RNA was isolated and used for quantitative PCR assay. Four to six animals were used for each stage after operation. , p<0.05; *, p<0.01 vs. normal tissue. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 2 Phase contrast image of tissue cultured Xenopus RPE1choroid on Day 14 and (A to H) detection of Xmmp9 and Xmmp18 expression on Day 5. Normally, 4 different zones can be discriminated around Day 14 of culture, depending on the cell–cell interactions. In the epithelial zone, both N-cadherin and Connexin-43 are detected, while in the transitional zone only N-cadherin can be detected (Nabeshima et al., 2013). (A–D) In situ hybridization of Xmmp9 and Xmmp18 at the early stage of culture. RPE cells migrating from the periphery of the explant (asterisks) and in the epithelial zone are positively stained for both Xmmp9 and Xmmp18. (E–H) No positive staining was detected when sense probes were applied. Cultures were treated for de-pigmentation before they were processed for in situ hybridization. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 3 Effects of MMP inhibitor, 1, 10-PNTL on RPE cell migration and neuronal differentiation. (A, B) In the control culture, RPE cells already began to migrate from the explants on Day 2 (arrow in A) and form the epithelial zone on Day 7. (C, D) In the presence of the inhibitor, only a few RPE cells were observed outside of the explant (arrows). Asterisks indicate tissue explants. (E– H) Neuronal differentiation was detected by bIII tubulin immunocytochemistry on Day 15. The inhibitor substantially suppressed differentiation of bIII tubulin-positive cells at 20 mM. (I) Cell counting of bIII tubulin-positive cells. The data are the mean cell number from 4 explants at each concentration of the inhibitor. Three separate experiments were performed and the representative data are shown in the figures. *, p<0.05 vs. cultured without the inhibitor (DMSO). [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 4 Effects of 1, 10-PNTL on the RPE cell-cell adhesion and Pax6 expression. Cell adhesion profiles were examined using anti-connexin-43 and N-cadherin antibodies. (A, B and E, F) In the control cultures, connexin-43 immunoreaction was detected in the epithelial zone, while N-cadherin was found both in the epithelial and transitional zones. (C, D and G, H) In the presence of the inhibitor, no positive staining for both antibodies was detected. (I–K) In the control culture, intense Pax6 immunoreactivity was detected in the transitional zone, while no positive cells were detected in the presence of the inhibitor (L–N). Asterisks indicate tissue explants and the dotted lines indicate the boundaries between the explant and the epithelial zone. Four separate experiments were performed and the representative data are shown in the figures. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5 Effects of 1, 10-PNTL on RPE cell proliferation. (A,B) In the control cultures, numerous cells in the transitional zone were labeled for BrdU. (C) By applying double immunostaining for BrdU (red color) and connexin-43 (green color), RPE cells that were labeled for BrdU were observed mostly in the transitional zone, while in the epithelial zone cells were stained for connexin-43 and were not labeled for BrdU. (D, E) In the presence of the inhibitor, only a few cells were labeled for BrdU. Asterisks indicate tissue explants and the dotted lines indicate the boundaries between the explant and the epithelial zone. Asterisks indicate tissue explants and the dotted lines indicate the boundaries between the explant and the epithelial zone. (F) Quantification of labeled cells for BrdU indicates that the inhibitor substantially inhibited RPE cell proliferation. The data are the mean cell number from four explants at each concentration of the inhibitor. For details of cell counting, see the methods section. Three separate experiments were performed and the representative data are shown in the figures. *, p<0.05 vs. cultured without the inhibitor. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6 Expression of TNF-a and IL-1b in the RPE and choroid at Day 1 after retinal removal (A), and expression of TNF-a and IL-1b, Xmmp9, and Xmmp18 in cultured tissues (B). (A) Twenty-four hours after retinal removal, both cytokine genes are markedly upregulated, indicating that inflammatory reaction had begun in the choroid. Three to four animals were used either for the control or Day 1 after retinal removal. (B) In the culture, expression of TNF-a and IL-1b is clearly observed on Day 3 and the expression level decreased considerably on Day 20. Expression of Mmp9 and Mmp18 exhibits a similar profile to that of cytokine genes. In the presence of 10 mM Withaferin A (W), expression levels of these genes on Day 3 substantially decreased. Three separate experiments were performed and representative data are shown in the figure. , p<0.05; *, p<0.01 vs. cultured at Day 0. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 7 Effects of the anti-inflammatory drugs dexamethasone (Dex) and Withaferin A on RPE cell migration and neuronal differentiation. (A–C) On Day 12 in the culture lacking Dex, RPE cells had migrated out from the periphery of the explant. The arrow indicates the distance between the periphery of the explant and the most outward RPE cells. Cells did not actively migrate when Dex was present in the culture at 50 mM (B) or 100 mM (C). (D, E) Similar effects by Withaferin A were observed on Day 8, and Withaferin A mostly suppressed RPE cell migration at 10 mM. Asterisks indicate tissue explants. (F) When neuronal differentiation was detected with ßIII tubulin immunostaining, Dex suppressed differentiation of ßIII tubulin-positive cells in a Dex-concentration-dependent manner. The data are the mean cell numbers from four explants at each concentration of the inhibitor. The results represent data from three separate experiments. , p<0.05; *, p<0.01 vs. cultured without Dex. [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 8 Summary of the hypothetical sequence of retinal regeneration in X. laevis. Surgical removal of the retina causes an inflammatory reaction in the capillary-rich tissue, the choroid, and some cytokines will be released in the tissue. These substances will induce RPE cells to upregulate Mmp genes. Accordingly, RPE cells detach from their basement membrane and migrate to the other basement membrane (the retinal vascular membrane), where they re-form an epithelial structure and finally upregulate the regeneration genes, Rax and Pax6. [Color figure can be viewed at wileyonlinelibrary.com]