REGULATION OF CYSTATIN S GENE EXPRESSION

哺乳动物的唾液腺产生分泌物，开始消化、洗澡和保护口腔。唾液半胱氨酸素是由颌下腺和腮腺分泌的蛋白质，被认为在这种保护中发挥了重要作用。半胱氨酸蛋白酶抑制剂是进化上保守的、自然产生的半胱氨酸蛋白酶抑制剂，它通过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来调节蛋白分解。尽管半胱氨酸蛋白酶在体外具有抑制作用，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胱抑素在唾液腺的生长、分化、形态发生和功能中的作用，我们建议研究在大鼠唾液腺中表达的胱抑素S基因的表达机制。这项建议将详细阐述我们以前的发现，即胱抑素S基因是独特的，因为它是细胞类型和唾液腺特异的，它在颌下腺出生后发育的特定阶段表达，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，ITS 5‘侧翼序列有三个唾液腺特异基因共有的序列元件(I、II和III)，插入在保守元件II和III之间的是GT富集区(GT富集区被认为抑制或增加特定基因的转录活性)。需要检验的两个主要或假说是：1)存在于所有唾液腺特异基因中的保守序列元件是决定唾液腺特异表型的遗传元件；2)这些保守序列元件以某种方式参与了唾液腺特异基因cystatin S表达的β-肾上腺素能调节。我们的具体目标是：1.确定保守的唾液腺特异dna序列元件在培养细胞中的作用2.鉴定和描述cystatin S启动子中调节ipr诱导的cystatin S基因表达的潜在调控元件3.验证顺式作用的保守的唾液腺特异调控元件，在组织培养中鉴定的介导ipr诱导的半胱氨酸氨基转移酶S基因表达的调控元件在转基因小鼠体内起作用4.确定伴随半胱氨酸氨基转移酶S基因表达变化的反式作用因子是否发生变化

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