Expression of keratin intermediate filament proteins is highly regulated during the development, growth, and differentiation of the epidermis. Our understanding of the function of these proteins and the network they assemble into is limited, though their role in the cell seems to be one other than purely structural. The broad, long-term objective of this proposal is to determine the functional significance of the keratins within epithelial cells and the role they have in the growth and differentiation of the epidermis.
The Specific Aims are 1) to determine how the expression of mutant keratin proteins in cultured epithelial cells affects cell growth, expression of cell differentiation, cell morphology, regulation of keratin synthesis and filament assembly, and 2) To determine how mutant keratin proteins expressed in the epidermis of transgenic mice affect in vivo epidermal growth parameters, expression of differentiation, and morphology of the epidermis. Both cultured cells and transgenic mouse model systems will be employed to create a comprehensive system that incorporates both flexibility and relevancy. Permanent cell lines will be created by stably transfecting epithelial cells with previously isolated deletion mutations of a human K14 epidermal keratin cDNA. Mutant K14 cDNAs will be expressed in cells using the dexamethasone inducible MMTV LTR promoter in order to achieve control of mutant expression and to isolate lines that express mutant keratins that may be lethal. The inducible expression of mutant keratins in cultured cells will enable study of the cellular response to an abnormal keratin network with respect to cell growth, differentiation, morphology, keratin synthesis and filament assembly. Expression of mutant K14 keratin in transgenic mouse epidermis will allow a direct assessment of the effect an abnormal keratin network has on the structure and integrity of the epidermis and on parameters of epidermal growth and differentiation. Keratin filament structure and organization are known to be altered in certain genetic diseases of the epidermis, thus, the expression of generically defective keratins which produce abnormal keratin networks in the transgenic mouse epidermis will provide a powerful system in which to evaluate the role of keratin networks in the function and structure of the epidermis. The effect of mutant keratin expression on cell growth will be determined by measuring cell doubling time and labeling indices. Whether mutant keratin expression affects the onset and progression of epidermal cell differentiation will be determined by immunolabeling and PAGE to detect differentiation specific proteins, counting cornified envelopes, and morphology. Morphology of cells expressing mutant keratin will be evaluated using light and electron microscopy in conjunction with immunolabeling. Previous studies suggest that expression of mutant keratin proteins inhibits keratin synthesis. To investigate this possibility, keratin synthesis will be measured on the transcriptional and translational levels in cans expressing abnormal keratin using Northern analysis and one- and two-dimensional PAGE.
