We have proposed that cytodifferentiation of the submandibular gland (SMG) acinar cells progresses by a sequence of integrated structural remodelling and biosynthetic steps involving qualitative and quantitative changes in the pattern of gene expression. We have defined the temporal sequencing and spatial coordination of many of the major events in SMG secretory cell cytodifferentiation including the appearance of beta-adrenergic receptors (beta AR) on the cell's surface. The mechanisms which regulate the sequence of transcriptional and post-transcriptional changes which reflect expression of the gene for secretory protein synthesis and the beta AR are not known. The proposed studies are designed to help explain some of the molecular mechanisms involved in the regulation of secretory cell cytodifferentiation and the appearance of the beta AR. Towards this goal we will perform tissue culture and related studies to evaluate the role of specific extracellular matrix components in the initiation and maintenance of secretory cell differentiation and isolate and clone cDNA for the beta AR and use this cDNA as a probe to isolate, clone and measure the developmental expression of the gene for the receptor.
The specific aims of the project are to: 1) Examine the effects of inhibition of basement membrane/extracellular matrix component deposition on SMG secretory cell differentiation, 2) construct a cDNA library from total SMG secretory cell poly(A)+ mRNA, 3) screen the library with antibodies or synthetic oligonucleotide probes to the beta AR to identify clones containing the cDNA for the receptor, 4) if necessary, use immunopurification of polysomes and/or hybridization selection procedures to obtain an mRNA fraction enriched for message to the beta AR and use this mRNA to produce an enriched cDNA library. Screen this library with antibodies or oligonucleotide probes to isolate the desired cDNA, 5) label the specific cDNA and use it as a probe to determine cytoplasmic content of specific mRNA during maturation of the gland as a measure of developmental expression of the and beta AR gene, 6) construct a SMG secretory cell total genomic library and use the cDNA probes to isolate and clone the gene for beta AR. These experiments will provide the knowledge base and molecular probes necessary to investigate the mechanisms regulating secretory cell cytodifferentiation and the expression of the beta AR. The information gained will be useful in understanding the chemical and structural basis of salivary gland neoplasms as well as a variety of diseases affecting salivary glands such as cystic fibrosis and Sjogren's Syndrome.
