Annual Report 2000This project investigates signal transduction pathways that regulate survival, growth, and differentiation in the lens and corneal epithelium. Recent studies have focused on the signaling pathway linking EGF receptor activation with c-fos transcription in the lens and on the functions of Cdk5, a member of the cyclin dependent kinase family, in both lens and corneal epithelium. Our previous studies of EGF-stimulated c-fos transcription in lens epithelial cells have demonstrated a requirement for 12(S)HETE, a lipoxygenase metabolite of arachidonic acid. Using a rabbit lens epithelial cell line, we have now shown that 12(S)HETE is required for activation of the classical isoforms of PKC, PKCalpha and PKCbeta2. In the absence of 12(S)HETE these PKC isoforms are not translocated to the membrane in response to EGF stimulation. We have further shown that the absence of 12(S)HETE has no effect on the ability of EGF to activate phospholipase Cgamma or the mitogen activated protein kinases, Erk1 and Erk2. Moreover, direct pharmacological inhibition of PKCalpha also blocks c-fos transcription without affecting EGF-stimulated signaling through Erk1,2. These findings demonstrate a role for 12(S)HETE in regulating PKC and suggest that simulataneous signaling through both PKC and mitogen activated protein kinases is necessary for EGF-stimulated c-fos transcription in lens epithelial cells. Our studies of Cdk5 suggest that this enzyme may regulate cell adhesion, migration, and survival in a variety of non-neuronal cells, including lens and corneal epithelial cells. Our previous immunofluorescence studies on rat cornea had indicated that Cdk5 is localized primarily along the basal aspect of the basal epithelial cells of the cornea. We have now expanded this study of Cdk5 localization to the mouse cornea and have demonstrated that the activator of Cdk5, p35, shows an overlapping pattern of localization. Moreover, Cdk5 and p35 co-immunoprecipitate from corneal epithelial cells, indicating that potentially active Cdk5/p35 complexes are present. Although we have been unable to measure Cdk5/p35 activity directly, gene transfer studies have provided indirect evidence that active Cdk5 may be present. Adenovirus vectors were used to overexpress either Cdk5 or a kinase inactive mutation, Cdk5T33, in a conditionally immortalized mouse corneal epithelial cell line, A6(1). The ability of the cells to adhere to fibronectin was increased by Cdk5 overexpression and decreased by Cdk5T33 overexpression. Since these two constructs differ by only a single amino acid, it seems likely that the difference in biological effect may be related to their difference in enzymatic activity. These in vitro results are paralleled by in vivo results obtained with transgenic mice expressing Cdk5 or Cdk5T33 under the control of the Aldh3 promoter. Of three transgenic lines overexpressing Cdk5, the line with the highest level of overexpression, 3.5 fold over endogenous Cdk5 levels, showed a significantly reduced rate of healing of a small debridement wound as compared to normal age-matched controls. In contrast, the rate of wound healing was not affected in a transgenic line with a similar level of Cdk5T33 overexpression. These findings suggest a biological role for Cdk5 in regulating corneal wound healing, possibly through regulation of cell-matrix interactions. Since Cdk5 is usually considered a neuron-specific enzyme, we have also explored its possible function in other selected non-neuronal cells in addition to lens and corneal epithelial cells. Studies in U373 astrocytoma cells have demonstrated a role for Cdk5 in regulating cell shape, adhesion, and apoptosis following heat shock. A role in apoptosis was also suggested in COS-1 cells, where expression of Cdk5, but not Cdk5T33, caused spontaneous apoptotic cell death. This cell line is being used to investigate the signaling pathway by which Cdk5 induces apoptosis.
Showing the most recent 10 out of 26 publications
