This project investigates cell cycle regulation in proliferating, quiescent, and differentiating lens cells through studies of proto-oncogenes, cyclins, and cyclin dependent kinases (Cdks). We have previously established that cyclin B/Cdc2 is present in embryonic rat lens fiber cells and that kinase activity associated with this complex peaks during denucleation of the primary lens fiber cells. To test whether cyclin B/ Cdc2 plays a causal role in fiber cell denucleation, we have used crystallin promoter constructs to overexpress proteins that will block Cdc2 activity in lens fiber cells. Analysis of lines overexpressing Wee1, a kinase that regulates Cdc2 activity, showed that expression of this enzyme was not effective in blocking Cdc2 activity in the lens. As an alternative approach, we have constructed a dominant negative mutation of Cdc2 under the control of the betaB1-crystallin promoter. Since this mutation is extremely effective in blocking cyclinB/Cdc2 activity in cultured cells and the betaB1-crystallin promoter gives very high levels of expression in developing lens fiber cells, this construct seems likely to be an effective inhibitor of cyclinB/Cdc2 activity in vivo. Transgenic mice bearing this construct should be available for analysis within a few months. Related studies have investigated the regulation of cyclin B and Cdc2 expression in differentiating lens fibers, since these genes are normally repressed in non-cycling cells. We have demonstrated that lens fiber cells lack p130/E2F-4 complexes, which normally repress transcription of the cdc2 gene in differentiating cells. We have also demonstrated that lens fiber cells contain other active Cdks, including Cdk7, Cdk8, and Cdk5, an enzyme that was previously thought to be brain specific. Expression of Cdk5 in lens fiber cells is particularly interesting, since this enzyme has recently been associated with programmed cell death of a variety of cell types during embryonic development. This observation suggests that Cdk5 may be responsible for apoptotic-like changes that occur during terminal differentiation of lens fiber cells. We are using several approaches to test this possibility. First, we have generated constructs of wild type Cdk5 or a dominant negative mutation of Cdk5 under the control of the betaB1-crystallin promoter to target expression to lens fiber cells in transgenic mice. Transgenic lines of both constructs have been obtained and are currently under analysis. Second, we are constructing adenoviral vectors bearing wild type Cdk5 or a dominant negative mutation of Cdk5. Adenoviral vectors are a highly effective means of transferring genes to differentiated cells in culture, which can be used in conjunction with differentiating explants of rat lens epithelia to explore the role of Cdk5 in lens differentiation. Finally, we are attempting to determine the substrates of Cdk5 in the lens by examining candidate proteins in vitro and by identifying proteins that are able to interact with Cdk5 in a yeast two-hybrid system. One potential substrate under investigation is alphaB-crystallin, which contains a conserved Cdk5 recognition sequence at a site that is phosphorylated in vivo. To identify other potential substrates we will soon begin screening a recently constructed embryonic rat lens cDNA library in yeast. In the course of studies on Cdk5 expression in the lens, we observed that this enzyme is also expressed in the developing and mature cornea. Further studies indicated that Cdk5 is expressed at especially high levels in the leading edge of migrating corneal epithelial cells during wound healing. A collaboration has been established with Dr. Joram Piatigorsky to express wild type and dominant negative Cdk5 in the corneal epithelium under control of the aldehyde dehydrogenase III promoter, which has recently been shown to target expression to this tissue. These studies will probe the biological role of Cdk5 in corneal development and wound healing. A second area of intense study is the regulation of proto-oncogene expression and cell proliferation in lens epithelial cells. We have demonstrated that 12(S)HETE, a lipoxygenase metabolite of arachidonic acid, is required by lens epithelial cells for c-fos transcription in response to growth factor stimulation. Studies of the mechanism of this effect have shown that 12(S)HETE is required for activation of protein kinase C (PKC). Further studies of this biochemical pathway will target specific steps in the signal transduction cascade using pharmacological compounds or adenoviral vectors bearing dominant negative constructs of essential enzymes. Because of the central role of PKC in the response of lens epithelial cells to 12(S)HETE, we have also examined the expression of protein 14-3-3, a known regulator of PKC activity. We have demonstrated that 14-3-3 is complexed with PKC in lens epithelial cells. Stable transfection of N/N1003 lens epithelial cells with 14-3-3 (eta isoform) significantly increased PKC activity. Experiments in progress will examine the isoform of PKC affected and the possible involvement of 12(S)HETE.
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