The major excreted protein (MEP) of mouse fibroblasts is the precursor to a lysosomal acid protease (cathepsin L) whose synthesis is induced by malignant transformation, growth factors, tumor promoters, and cAMP. Cathepsin L is produced both by normal cells and tumors and may function in exerting the local extracellular and distal humoral effects of some tumors. Cathepsin L plays a major role in intracellular protein catabolism and has been implicated in immune system, myofibrillar, myocardial, and renal pathology, as well as in bone resorption and arthritis. Understanding the regulatory mechanisms involved in the expression of MEP/cathepsin L may yield insight into the biology of both normal and transformed cells, and also provide a basis for designing therapeutic strategies which may reduce the morbidity in those populations, especially the elderly, that are affected by cancer, osteoporosis, and some forms of arthritis and kidney disease. Furthermore, the regulation of MEP gene expression is unlike other systems being studied.Phorbol ester stimulation of the MEP promoter requires new protein synthesis and the presence of regions downstream of the transcription initiation site. This response to TPA is not mediated by a classical enhancer or a mechanism involving attenuation or termination of transcription. Therefore, the isolation of the mouse MEP promoter and eventually its human counterpart will permit us to directly study the novel transcriptional regulation of this growth factor- and transformation-sensitive gene. To address these objectives, mutational analysis of both mouse and human MEP-reporter gene plasmids and nuclear factor binding studies will be performed to identify the regions responsible for the regulation of MEP gene expression. These studies will be accompanied by mapping of the intron/exon structure of the mouse and human MEP genes. Furthermore, immunoprecipitation, RNA slot blot, and nuclear run-off assays will be utilized to assess the regulation of MEP expression by a variety of signal transducing agents in fibroblasts, macrophages, and renal cells.
Specific Aim 1. Identify the upstream and downstream cis-acting sequence motifs responsible for the regulation of expression of the MEP gene.
Specific Aim 2. Identify, isolate, and characterize the human MEP promoter.
Specific Aim 3. Map the intron/exon structure of both the mouse and human MEP genes and sequence the complete upstream and first intron regions.
Specific Aim 4. Assess the effect on MEP expression of tumor promoters, growth factors, second messengers, hormones, and cytokines in a variety of mouse and human cells lines, including fibroblasts, macrophages, and renal cells.
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