The function of Dkk3 in the ocular lens

Abstract

Cataract, the clouding of the lens, is the leading cause of blindness worldwide. While cataract surgery (CS) is effective in treating cataract, it does have side effects. After CS, the residual lens epithelial cells (LECs) migrate to the posterior part of the capsule and cause posterior capsular opacification (PCO). PCO is caused by fibrotic EMT “(“Epithelial to Mesenchymal Transition”).” The canonical TGF-β signaling pathway is well-known as the main driver of fibrotic EMT in most cell types, including LECs post cataract surgery (PCS). While TGF-β signaling is important, the mechanisms by which this pathway is activated PCS are poorly understood. In other systems, canonical Wnt signaling synergizes with canonical TGF-β signaling to drive fibrotic EMT. A Wnt reporter (Wnt R) mouse line has been used to investigate the canonical Wnt signaling in LECs. While canonical Wnt signaling is inactive in the naïve lens, it starts upregulating at 12 hr PCS and has a robust upregulation at 48 hr PCS. Canonical TGF-β signaling starts to upregulate at 48 hr PCS. This leads to the global hypothesis that canonical TGF-β and canonical Wnt signaling work in synergy to drive the fibrotic response PCS. The Dickkopf family (Dkks) are well-known inhibitors of canonical Wnt signaling with five family members: Dkk1, Dkk2, Dkk3, Dkk4, and Soggy. Unlike other Dkks that inhibit canonical Wnt signaling, Dkk3 plays different roles in regulating canonical Wnt signaling, depending on the cell type. Since Dkk3 is one of the most expressed genes in the mouse lens epithelium, a mouse model with a germline deletion of the Dkk3 gene (Dkk3 -/-) was used to study the role of Dkk3 in the eye, particularly in lens epithelium. This study found that canonical Wnt signaling is prevented in Dkk3 -/- Wnt R -/+ LECs at both 0 hr and 48 hr PCS. Moreover, it was shown that adding exogenous Dkk3 to the lens during the surgery blocks canonical Wnt signaling post-surgery. These were unexpected in that Dkk3 removal/inhibition, in turn, inhibits Wnt, which goes against Dkk’s known role as a Wnt inhibitor. The contradictory results proposed that biological effects of where in the cell Dkk3 interacts with its receptor, Kremen-1, may differ. Thus, I investigated the levels of Kremen-1 in wild-type (WT) and Dkk3 -/- LECs and its localization in the lens cells, and the results have shown that: 1) Kremen-1 levels are significantly higher in the WT LECs than Dkk3 -/-, 2) While Kremen-1 is located in the membrane portion of the cells, it is primarily localized in the secretory pathway, specifically in the endoplasmic reticulum (ER), 3) The intensity levels of Wheat Germ Agglutinin (WGA) staining, which is used as a cell membrane marker, are higher in the WT LECs than in Dkk3 -/-. The above results have suggested that intracellular Dkk3 can interact with intracellular Kremen-1, stabilizing it and preventing its degradation or endocytosis while preventing Kremen-1 from transporting to the cell surface and becoming membrane-bound, letting LRP5/6 interact with Wnt ligands, activating canonical Wnt signaling. In contrast, when Dkk3 is outside the cell, it can interact with cell surface Kremen-1 and LRP5/6, inhibiting canonical Wnt signaling. In conclusion, these results and data suggest that the biological consequences of Dkk3’s interaction with Kremen-1 vary and have different effects on modulating canonical Wnt signaling depending on its location in the lens cells.