REGULATION OF CYSTATIN S GENE EXPRESSION

哺乳动物的唾液腺分泌的分泌物可以促进消化、沐浴和保护口腔。唾液胱抑素是由下颌骨和腮腺分泌的蛋白质，被认为在这种保护中起主要作用。半胱氨酸抑制素是一种进化上保守的天然半胱氨酸蛋白酶抑制剂，可调节内源性半胱氨酸蛋白酶以及微生物病原体和某些病毒的蛋白酶的蛋白水解。虽然半胱氨酸抑制素在体外抑制半胱氨酸蛋白酶，但其在体内的功能尚未被详细描述。由于我们的长期目标是了解半胱抑素在唾液腺生长、分化、形态发生和功能中的作用，我们建议研究半胱抑素S基因表达的调控机制，该基因在大鼠唾液腺中表达。本提案将详细阐述我们之前的发现，胱氨酸抑制素S基因的独特之处在于它是细胞类型和唾液腺特异性的，它在出生后颌下腺发育的特定阶段表达，它在成年动物中被关闭，并且可以通过β受体介导的机制在成年大鼠中诱导。此外，其5'侧翼序列具有唾液腺特异性基因共有的三个序列元件（I、II和III），并且在保守元件II和III之间插入了一个富含GT的区域（富含GT的区域被认为可以抑制或增加特定基因的转录活性）。需要验证的两个主要假设是：1)存在于所有唾液腺特异性基因中的保守序列元件是决定唾液腺特异性表型的遗传元件；2)这些保守序列元件以某种方式参与唾液腺特异性基因胱抑素s表达的β -肾上腺素能调节。确定涎腺特异性DNA序列元件在培养细胞中的作用2。鉴定和描述胱氨酸抑制素S启动子中介导知识产权诱导的胱氨酸抑制素S基因表达的潜在调控元件利用转基因小鼠验证在组织培养中鉴定的顺式作用的保守的唾液腺特异性调控元件和介导ipr诱导的胱抑素S基因表达的调控元件在体内起作用4。确定反式作用因子是否随胱抑素S基因表达的变化而发生变化

Mammalian salivary glands produce secretions that initiate digestion and bathe and protect the oral cavity. Salivary cystatins, proteins secreted by the submandibular and parotid glands, are believed to play a major role in this protection. Cystatins are evolutionarily conserved, naturally occurring cysteine proteinase inhibitors that regulate proteolysis by endogenous cysteine proteinases, as well as by proteinases of microbial pathogens and of some viruses. Although cystatins inhibit cysteine proteinases in vitro, their in vivo functions have not been delineated in detail. Since our long-term goal is to understand the role of cystatins in growth, differentiation, morphogenesis, and function of salivary glands, we propose to examine mechanisms governing expression of the cystatin S gene, which is expressed in rat salivary glands. This proposal will elaborate upon our previous findings that the cystatin S gene is unique in that it is cell type-and salivary gland-specific, it is expressed at a specific stage of posnatal development of the submandibular gland, it is turned off in adult animals, and it can be induced in adult rats by beta-receptor mediated mechanisms. In addition, its 5' flanking sequence has three sequence elements (I, II, and III) that are common to salivary gland-specific genes, and inserted between conserved elements II and III is a GT rich region (GT rich regions are thought to either inhibit or increase transcriptional activity of specific genes). Two major or hypotheses to be tested are that: 1) the conserved sequence elements present in all salivary gland-specific genes are the genetic elements that dictate salivary gland-specific phenotype, and 2) these conserved sequence elements somehow participate in the beta-adrenergic modulation of expression of a salivary gland-specific gene, cystatin S. Our specific aims are to: 1. determine the role of the conserved salivary gland-specific DNA sequence elements in cells in culture 2. identify and delineate potential regulatory elements In the cystatin S promoter that mediate IPR-induced cystatin S gene expression 3. verify that the cis-acting conserved salivary gland-specific regulatory elements, and the regulatory elements that mediate IPR-induced cystatin S gene expression, that were identified in tissue culture act in vivo using transgenic mice 4. determine whether there are changes in trans-acting factors that accompany changes in expression of the cystatin S gene