ABSTRACT
BACKGROUND: The vitreous has been shown to induce epithelial-mesenchymal transdifferentiation because it induces fibroblast-like morphology, enhanced migration and invasion in retinal pigment epithelial cells in proliferative vitreoretinopathy. Rac1 is the principal mediator of cell migration. In the current study, the relationship between Rac1 and cell migration, and invasion in vitreous-transformed retinal pigment epithelial cells was investigated using NSC23766, a specific inhibitor of Rac guanosine-5'-triphosphatase activity, and the involvement of a Rac1 guanosine-5'-triphosphatase-dependent pathway was detected. DESIGN: One-way design with multiple levels and repeated measurement design. PARTICIPANTS AND SAMPLES: The vitreous humor was collected from 20 healthy donor eyes and the retinal pigment epithelial cells were obtained from 9 healthy donor eyes. METHODS: Human low-passage retinal pigment epithelial cells were treated with normal medium or 25% vitreous medium. Rac1 activity was measured using a pull-down assay. The cytotoxicity of NSC23766 was measured using the trypan blue dye exclusion test. Cell migration was measured using a wound healing assay. Cell invasion was determined using a transwell invasion assay. Protein expression of Rac1 and phosphorylation of LIM kinase 1 and cofilin were detected by Western blot analysis. MAIN OUTCOME MEASURES: Cell migration, invasion, Rac1 activity and phosphorylation of LIM kinase 1 and cofilin. RESULTS: Rac1guanosine-5'-triphosphatase was activated in vitreous-transformed retinal pigment epithelial cells. A Rac inhibitor suppressed vitreous-induced migration and invasion in retinal pigment epithelial cells. Cofilin phosphorylation was activated by vitreous treatment but blocked by NSC23766. CONCLUSIONS: Rac1 mediates vitreous-transformed retinal pigment epithelial cells' plasticity of mesenchymal movement via Rac1 guanosine-5'-triphosphatase-dependent pathways that modulate LIM kinase 1 and cofilin activity. Rac inhibition may be considered a novel treatment for proliferative vitreoretinopathy.
Subject(s)
Cell Movement/physiology , Mesenchymal Stem Cells/cytology , Retinal Pigment Epithelium/cytology , Vitreoretinopathy, Proliferative/pathology , Vitreous Body/physiology , rac1 GTP-Binding Protein/physiology , Adolescent , Adult , Aged , Aminoquinolines/pharmacology , Blotting, Western , Cell Line, Transformed , Cell Survival , Cell Transdifferentiation , Cells, Cultured , Enzyme Inhibitors/pharmacology , Humans , Lim Kinases/metabolism , Mesenchymal Stem Cells/metabolism , Middle Aged , Phosphorylation , Pyrimidines/pharmacology , Retinal Pigment Epithelium/metabolism , Young Adult , rac1 GTP-Binding Protein/antagonists & inhibitorsABSTRACT
The recombinant eukaryotic expression vector pCMV4-rmCGbeta, inserted full-length cDNA of the beta-subunit of rhesus monkey chorionic gonadotropin (rmCGbeta), as DNA immuno-contraceptive against CGbeta glycoprotein, has previously demonstrated the biological expression of rmCGbeta in vitro and in vivo. The plasmid DNA of pCMV4-rmCGbeta was inoculated into BALB/c mice at different doses and routes as DNA immuno-contraceptive to understand its antifertility effect. The results of immune responses indicated that the intradermal inoculation is the optimal pCMV4-rmCGbeta DNA delivery method for BALB/c mice, and the dose of 10 microg should be enough to elicit immune response. With different doses from 10-50 microg, marked reductions in the fertility of the female mice after two intramuscular inoculations of pCMV4-rmCGbeta DNA were seen, while the similar level of humoral immune responses were induced. With the dose of 20 microg of pCMV4-rmCGbeta DNA, the mice showed reduction in fertility from intraperitoneal, and intradermal to intramuscular inoculating method. The antifertility effect of antiserum from immunized mice confirmed that the antibodies elicited by pCMV4-rmCGbeta DNA could prevent pregnancy in female mice. At the same time, the full-length cDNA of beta-subunit of mouse chorionic gonadotropin (muCGbeta) was cloned from placenta and sequenced for the first time (GenBank Accession No. AF333067). Sequence analysis showed that muCGbeta shares 99.6% homology with rmCGbeta and 90.6% with hCGbeta respectively. The results indicated that the infertility of BALB/c mice induced by pCMV4-rmCGbeta contraceptive should be further studied as a CGbeta DNA contraceptive. (Mol Cell Biochem 231: 89-96, 2002)
