Regulatory role of PI3K signaling pathways in lens differentiation and function

Thomas Jefferson University, Philadelphia PA

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Abstract

Project Summary/Abstract The process of creating the lens, a tissue whose function of focusing images on the retina depends on its transparency, involves a complex coordination of signaling pathways. This proposal is aimed at discovering new molecular aspects of the signaling pathways that our studies have shown regulate three major aspects of lens differentiation: 1) the decision of lens epithelial cells to withdraw from the cell cycle and initiate their differentiation program, 2) the removal of non-nuclear organelles from differentiating lens fiber cells, and 3) the spatiotemporal elimination of fiber cell nuclei. We build on our discovery that PI3K signaling pathways have essential functions in regulating each of these stages of lens differentiation, with investigations that will determine the key downstream effector pathways of PI3K that make it possible for this signaling molecule to mediate these distinct steps in the development of the lens. Despite the links of FOXO transcription factors to the expression of many molecular regulators of the lens differentiation program, FOXO function in lens development has not been investigated. Our studies now provide the first evidence that FOXO1 and FOXO4 are expressed in the developing lens, and that blocking the PI3K/Akt signaling axis induces their translocation to the nucleus and transcriptional activation. In addition, the literature shows that these FOXO transcription factors are key transcriptional regulators of the expression of p27, a cell cycle inhibitor we previously linked to lens epithelial cell initiation of differentiation, the expression of Beclin-1 and LC3B, key molecules in the autophagy mechanism that mediates the removal of non-nuclear organelles from differentiating lens fiber cells, and the expression of Bim-1 and FasL, both of which can function in the activation of caspase-3, which we have recently shown plays multiple roles in the process of eliminating nuclei from lens fiber cells. In Aim 1, we present studies that investigate the roles of FOXO transcription factors in the withdrawal of lens epithelial cells from the cell cycle to initiate their differentiation program and in the elimination of organelles during lens development. While blocking all PI3K downstream effector pathways induces removal of fiber cell nuclei, we discovered that suppressing Akt, which induces loss of its non-nuclear organelles, is not alone responsible for nuclear removal. In Aim 2, we will identify the different PI3K signaling effectors involved in nuclear elimination, and the specific steps in the removal of nuclei that they regulate. These studies will examine the requirement for inactivation of the PI3K downstream effector Rac in this process. Our preliminary studies have implicated suppression of Rac in the pathway required for condensation of fiber cell nuclei. We will also examine the link between PI3K inactivation and activation of caspase-3, which we showed induces DNA cleavage events and fragmentation of the nuclear lamina that are required for nuclear removal. And finally, we include studies that will determine if ERK is the survival signal that protects fiber cells during the process of nuclear elimination.