Qingxian Lu

Taz-Tead1 Links Cell-Cell Contact to Zeb1 Expression, Proliferation, and Dedifferentiation in Retinal Pigment Epithelial Cells

PURPOSE. The Hippo signaling pathway imposes the cell contact inhibition that establishes organ size and tissue topology from Drosophila to mammals. This pathway regulates activity of the Yap and Taz transcription factors, which link epithelial-mesenchymal transition (EMT) to cell proliferation. Here, the authors provide evidence that Taz and its coactivator, Tead1, regulate expression of the EMT transcription factor Zeb1 to control RPE cell proliferation and differentiation. METHODS. Real-time PCR was used to examine mRNA expression during RPE dedifferentiation in primary cultures of RPE cells and after knockdown of Yap and Taz by lentivirus shRNA. Immunofluorescence was used to follow subcellular localization of proteins in cells. Chromatin immunoprecipitation was used to detect Taz at the Zeb1 promoter in vivo. RESULTS. Zeb1 is overexpressed during RPE dedifferentiation, leading to cell proliferation, EMT, and repression of the RPE specification transcription factor gene Mitf. Taz-TEAD1 translocation to the nucleus coincides with loss of cell-cell contact and with onset of Zeb1 expression in the nucleus. shRNA knockdown of Taz prevented the overexpression of Zeb1 and, in turn, prevented proliferation, repression of Mitf and Mitf target genes, and EMT when RPE cells were placed in primary culture. Taz binds to the Zeb1 promoter in vivo, suggesting that it directly induces Zeb1 transcription. CONCLUSIONS. These results provide evidence of a molecular mechanism linking cell-cell contact to cell proliferation and dedifferentiation in RPE cells.

Exosomes from Retinal Astrocytes Contain Antiangiogenic Components That Inhibit Laser-induced Choroidal Neovascularization

Background: It is unknown whether the exosomes derived from neurosensory retinal cells regulate angiogenesis in the eye. Results: Exosomes from retinal astrocytes contain multiple antiangiogenic components that inhibit laser-induced choroidal neovascularization. Conclusion: Exosomes from monolayer culture of retinal astrocytes, but not retinal pigment epithelium, are antiangiogenic. Significance: Retinal astrocyte exosomes might be a potential for clinical applications in the treatment of choroidal neovascularization. Exosomes released from different types of host cells have different biological effects. We report that exosomes released from retinal astroglial cells (RACs) suppress retinal vessel leakage and inhibit choroidal neovascularization (CNV) in a laser-induced CNV model, whereas exosomes released from retinal pigmental epithelium do not. RAC exosomes inhibit the migration of macrophages and the tubule forming of mouse retinal microvascular endothelial cells. Further, we analyzed antiangiogenic components in RAC exosomes using an angiogenesis array kit and detected several endogenous inhibitors of angiogenesis exclusively present in RAC exosomes, such as endostatin. Moreover, blockade of matrix metalloproteinases in the cleavage of collagen XVIII to form endostatin using FN-439 reverses RAC exosome-mediated retinal vessel leakage. This study demonstrates that exosomes released from retinal tissue cells have different angiogenic effects, with exosomes from RACs containing antiangiogenic components that might protect the eye from angiogenesis and maintain its functional integrity. In addition, by identifying additional components and their functions of RAC exosomes, we might improve the antiangiogenic therapy for CNV in age-related macular degeneration and diabetic retinopathy.

14-3-3σ and p63 play opposing roles in epidermal tumorigenesis

14-3-3σ plays a regulatory role in epidermal epithelial differentiation and loss of 14-3-3σ leads to increased proliferation and impaired differentiation. A tumor suppressor function for 14-3-3σ has been proposed based on the fact that some epithelial-derived tumors lose 14-3-3σ expression. p63, a p53 family member, is a master regulator of epidermal epithelial proliferation and differentiation and is necessary for the epidermal development. The function of p63 in tumorigenesis is still controversial and poorly defined as multiple isoforms have been found to play either collaborative or opposing roles. By using repeated epilation heterozygous (Er/+) mice containing a dominant-negative 14-3-3σ mutation, the functional relationship of p63 with 14-3-3σ in epidermal proliferation, differentiation and tumorigenesis was investigated. It was found that p63, particularly the ΔNp63α isoform, was strongly expressed in 14-3-3σ-deficient keratinocytes and knockdown of p63 remarkably inhibited proliferation in these cells. To study the functional roles of 14-3-3σ and p63 in epidermal tumorigenesis, we adopted a 7,12-dimethylbenzanthracene/12-O-tetradecanoyl-phorbol-13-acetate (DMBA/TPA) two-stage tumorigenesis procedure to induce formation of skin papillomas and squamous cell carcinomas in Er/+ mice and identified strong p63 expression in resultant tumors. The loss of one allele of p63 caused by the generation of Er/+/p63(+/-) double compound mice decreased the sensitivity to DMBA-/TPA-induced tumorigenesis as compared with Er/+ mice. This study shows that p63 and 14-3-3σ play opposing roles in the development of skin tumors and that the accumulation of p63 is essential for Ras/14-3-3σ mutation-induced papilloma formation and squamous cell carcinoma carcinogenesis.

TAM Receptor Knockout Mice Are Susceptible to Retinal Autoimmune Induction

PURPOSEnTAM receptors are expressed mainly by dendritic cells and macrophages in the immune system, and mice lacking TAM receptors develop systemic autoimmune diseases because of inefficient negative control of the cytokine signaling in those cells. This study aims to test the susceptibility of the TAM triple knockout (tko) mice to the retina-specific autoantigen to develop experimental autoimmune uveoretinitis (EAU).nnnMETHODSnTAM tko mice that were or were not immunized with interphotoreceptor retinoid-binding protein (IRBP) peptides were evaluated for retinal infiltration of the macrophages and CD3(+) T cells by immunohistochemistry, spontaneous activation of CD4(+) T cells, and memory T cells by flow cytometry and proliferation of IRBP-specific CD4(+) T cells by [(3)H]thymidine incorporation assay. Ocular inflammation induced by IRBP peptide immunization and specific T cell transfer were observed clinically by funduscopy and confirmed by histology.nnnRESULTSnTko mice were found to have less naive, but more activated, memory T cells, among which were exhibited high sensitivity to ocular IRBP autoantigens. Immunization with a low dose of IRBP and adoptive transfer of small numbers of IRBP-specific T cells from immunized tko mice caused the infiltration of lymphocytes, including CD3(+) T cells, into the tko retina.nnnCONCLUSIONSnMice without TAM receptor spontaneously develop IRBP-specific CD4(+) T cells and are more susceptible to retinal autoantigen immunization. This TAM knockout mouse line provides an animal model with which to study the role of antigen-presenting cells in the development of T cell-mediated uveitis.

HMGB1 is an early and critical mediator in an animal model of uveitis induced by IRBP‐specific T cells

It is largely unknown how invading autoreactive T cells initiate the pathogenic process inside the diseased organ in organ‐specific autoimmune disease. In this study, we used a chronic uveitis disease model in mice—EAU—induced by adoptive transfer of uveitogenic IRBP‐specific T cells and showed that HMGB1, an important endogenous molecule that serves as a danger signal, was released rapidly from retinal cells into the ECM and intraocular fluid in response to IRBP‐specific T cell transfer. HMGB1 release required direct cell–cell contact between retinal cells and IRBP‐specific T cells and was an active secretion from intact retinal cells. Administration of HMGB1 antagonists inhibited severity of EAU significantly via mechanisms that include inhibition of IRBP‐specific T cell proliferation and their IFN‐γ and IL‐17 production. The inflammatory effects of HMGB1 may signal the TLR/MyD88 pathway, as MyD88−/− mice had a high level of HMGB1 in the eye but did not develop EAU after IRBP‐specific T cell transfer. Our study demonstrates that HMGB1 is an early and critical mediator of ocular inflammation initiated by autoreactive T cell invasion.

14-3-3σ Controls Corneal Epithelium Homeostasis and Wound Healing

PURPOSEnTo investigate the functional role of 14-3-3σ in regulation of the corneal epithelial proliferation, differentiation, and wound-healing response.nnnMETHODSnCorneal phenotypes were investigated in heterozygous repeated epilation (Er) mice carrying mutations in the sfn (14-3-3σ) gene. Immunohistochemistry was used to study the corneal morphogenesis of the Er/Er embryos at embryonic day (E)18.5. Corneal homeostasis and the wound-healing response were investigated macroscopically and microscopically in the adult heterozygous Er mice. Corneal epithelial cell proliferation and differentiation were assessed by BrdU incorporation and immunohistochemistry with specific antibodies for differentiation markers. Furthermore, corneal stroma neovascularization and meibomian gland degeneration were examined by immunohistochemistry. The healing of corneal wounds after debridement was monitored and visualized by fluorescent staining.nnnRESULTSnHomozygous mutation of 14-3-3σ led to defects in embryonic corneal epithelial development and differentiation, whereas young heterozygotes showed normal corneal development and homeostasis. However, older heterozygotes displayed a dramatic corneal wound-healing defect characterized by hyperplastic basal progenitor cells (some of which undergo a differentiation switch to express markers of keratinized epidermis); cornea stroma changes including neovascularization; and corneal opacity, leading to plaque formation. Aged heterozygotes also showed meibomian gland atrophy.nnnCONCLUSIONSn14-3-3σ is essential for corneal epithelium differentiation, and plays an important role in corneal epithelium development and daily renewal of the adult corneal epithelium.