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C.M. Garcia, D.C. Beebe; Signaling Through TGFßRII Is Not Required for, but Contributes to Secondary Cataract Formation in vitro and in vivo . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4641.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Secondary cataracts are formed by the transdifferentiation of lens epithelial cells into myofibroblasts. TGFß signaling has been implicated as an important regulator of myofibroblast formation in the lens and other organs. We demonstrated that lenses develop normally in the absence of the sole type II TGFß receptor (TGFßRII; Beebe, et al. Int J Dev Biol. 2004;48:845–56). We used these knockout lenses to measure the extent of lens myofibroblast formation in the absence of TGFßRII signaling in vitro and in vivo. Methods: LeCre mice, in which the Pax6 ectoderm enhancer drives the expression of Cre recombinase in the lens, were mated to mice that had exon 2 of the TGFßRII gene flanked by loxP sequences. Progeny were genotyped using PCR. Lens explants were harvested immediately post mortem or cultured for up to 4 days in basal medium or medium supplemented with TGFß2. Cataract surgery was performed on mice to follow the formation of secondary cataracts in vivo. Tissues were analyzed by western blotting or immunocytochemistry using antibodies to α–smooth muscle actin (αSMA), other myofibroblast markers, or molecules downstream of TGFßRII signaling (pSmad2 and Smad4). Results: Lens epithelial cells expressed low levels of αSMA at the time of explantation. After four days in defined medium, the levels of αSMA increased greatly in TGFßRIIfx/fx Cre– (wild type) and Cre+ (knockout) mice, although slightly lower levels of αSMA accumulated in knockout epithelial cells. Similar results were seen following cataract surgery. Wild type lens cells had little pSmad2 or Smad4 nuclear staining at explantation (T0) or after 4 hours in culture. Treatment with TGFß2 increased pSmad2 and Smad4 staining in the nucleus. In contrast, TGFßRII knockout lens explants had more nuclear pSmad2 and Smad4 staining than wild type explants at T0 or after 4 hours of culture. Treatment of knockout epithelial cells with TGFß2 caused the loss of pSmad2 and Smad4 staining in the nucleus and cytoplasm. Future experiments will be designed to analyze alternative signaling pathways for TGFß2 that are independent of TGFßRII. Conclusions: Signaling through TGFßRII is not required for myofibroblast transdifferentiation from lens epithelial cells in vivo or in vitro. Other pathways must contribute to myofibroblast formation in the lens. Deletion of TGFßRII uncovers another TGFß signaling pathway. Through this pathway, TGFß2 can inhibit Smad signaling.
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