Signals Involved in Lens Regeneration a Thrombin Activated Protein

As in other wounded tissues, serum clotting factors promote hemostasis when the pigmented epithelium is wounded by lentectomy. A major discovery was that thrombin is required to induce PECs of the newt dorsal iris cells to re-enter the cell cycle (Imokawa and Brockes, 2003). One of the first events in lens regeneration is the selective transient upregulation of active thrombin in the dorsal iris within 20-30 minutes (figure 3.15). Inactivation of thrombin by the inhibitors PPACK or antithrombin III resulted in failure of PECs to re-enter the cell cycle. The adult newt and the axolotl both regenerate their limbs, but the axolotl cannot regenerate its lens. Thrombin is upregulated after limb amputation in both newt and axolotl (see Chapter 14), but not after lentectomy in the axolotl, further strengthening the evidence that thrombin plays a key role in initiating newt lens regeneration (Imokawa and Brockes, 2003).

FIGURE 3.15 (A) Expression of thrombin activity in the dorsal iris (Di) but not the ventral iris (Vi) after lentectomy, as visualized by overlaying a section with a fluorogenic thrombin substrate. (B) A similar section treated with the thrombin inhibitor PPACK. No reaction is now visible in the dorsal iris. Pigmented epithelial cells fail to re-enter the cell cycle in PPACK-treated lentectomized eyes. Scale bar = 100 |m. Reproduced with permission from Imokawa and Brockes, Selective activation of thrombin is a critical determinant for vertebrate lens regeneration. Curr Biol 13:877-881. Copyright 2003, Elsevier.

newt dorsal iris following lentectomy. Lentectomy releases prothrombin (PT) and an as yet unidentified inactive plasma protein (Fi) in both the dorsal (Di) and ventral (Vi) iris. Tissue factor (TF) is expressed only dorsally, where it acts with clotting factors to convert prothrombin to thrombin (T, red). Thrombin then converts Fi to its active form (Fa). Fa induces DNA synthesis of pigmented epithelial cells in the dorsal iris.

newt dorsal iris following lentectomy. Lentectomy releases prothrombin (PT) and an as yet unidentified inactive plasma protein (Fi) in both the dorsal (Di) and ventral (Vi) iris. Tissue factor (TF) is expressed only dorsally, where it acts with clotting factors to convert prothrombin to thrombin (T, red). Thrombin then converts Fi to its active form (Fa). Fa induces DNA synthesis of pigmented epithelial cells in the dorsal iris.

Thrombin itself is produced through the activation of prothrombin by tissue factor. Prothrombin is released from damaged vasculature in the lentectomized newt eye, but there is no marked dorsoventral difference in the vascular density of the iris that could account for the selective activation of prothrombin dorsally. Imokawa and Brockes (2003) have therefore hypothesized that tissue factor is selectively released dorsally; the resulting thrombin then activates a protein that triggers PEC re-entry into the cell cycle (FIGURE 3.16), as has been shown for the regenerating newt limb (Tanaka and Brockes, 1999; Chapter 14). This hypothesis remains to be tested.

Cell cycle re-entry and proliferation of dorsal iris PECs appears to involve downregulation of the cell-cycle inhibiting retinoblastoma (Rb) protein, not by hypophosphorylating it as in other cells (Thitoff et al., 2003). As the new lens forms, Rb expression is upregu-lated again in the differentiating lens fibers, but not in the lens epithelium. The PECs of lentectomized newts immersed in solutions of the cyclin-dependent kinase 2-selective inhibitor, SU9516, were able to dedifferenti-ate and form a small lens vesicle, but the cells of the vesicle were unable to proliferate to form a new lens (Tsonis et al., 2004). This result suggests that dedifferentiation and cell cycle re-entry by PECs are not mechanistically linked. Two members of the complement system, C3 and C5, have been shown to stimulate mitosis of monocytes and B-cells at low concentration in vitro and may play a similar role in the proliferation of cells in the regenerating lens vesicle of the newt (Kimura et al., 2003). C3 is strongly expressed in the stroma and the PECs of the newt iris after lentectomy, and C5 is expressed in the regenerating lens vesicle.

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