HEPARIN CHEMISTRY REGULATING LIVER GENE EXPRESSION

肝脏特异性基因的表达，特别是它们的mRNA合成， 发现依赖于肽激素和肝素的协同作用 （HPs）。 比较超过40+种完整的、原生的 糖胺聚糖（GAG）和PG表明，只有HP或PGS与HP- 与GAG链一样，可以与肽激素合作调节mRNA 组织特异性基因的合成。 相比之下，硫酸肝素（HS）， 在所有浓度和所有来源，证明无效，甚至 抑制性的 所有测试的PG均观察到转录后效应， 和糖胺聚糖，与所有种类的HP和较弱的硫酸皮曼坦 （DS）。 PG在以下方面的效力等级顺序 转录后效应为HP-PGs>> HS-PGs>> DS-PGs>>软骨素 硫酸盐-PG（CS-PG）和活性GAG中，HP> DS。 我们建议完成结构-功能分析，持续进行，以确定 活性PG和GAG的化学性质，使它们能够调节基因 表情 我们完成的研究使我们能够专注于HP， HP-PGs的生物活性成分，PG中最活跃的种类 能够调节肝细胞中的基因表达。 HS密切相关 化学上无活性，将用作阴性对照。 的 所有记录为HP敏感的生物测定将：[1] 检测核形状变化的形态学测定;] 2）分子 mRNA合成和胰岛素应答性细胞丰度的杂交测定 基因;和3）间隙连接的电生理学测定。 细胞将被 当在无血清培养基中时，用测试HP或HP组分处理 仅补充完全定义和纯化的激素或生长 因子、为每个基因量身定制的组成和浓度。 的 分析的形式是：1）完成HP和 具有HP样活性的聚阴离子（例如苏拉明），使我们能够识别 特别是活跃的物种; 2）解剖最活跃的物种， 分子量（链长）、电荷密度、度 硫酸化的形式和抗凝活性的程度;[3]化学上 并从结构上分析最活跃的HP中最活跃的部分 和聚阴离子; 4）从化学分析中推断出活性物质 分数有共同点，并相应地准备修改版本; 5） 评估修饰形式的生物活性和6）重复3 - 5 直到鉴定出活性糖序列。] 鉴定的最具活性的HP糖将用于启动 HP/胰岛素信号转导机制的研究 基因表达的调控。 我们将首先集中研究 钙和细胞内pH值，因为我们以前的研究表明， 一种已知的第二信使cAMP的膜永久性类似物， 不能与HP协同调节转录速率， 检测任何肝脏特异性基因。 [We还将评估HP是否会修改 胰岛素受体水平或胰岛素对其自身的结合亲和力 受体或IGF I或II的受体。

Expression of liver-specific genes, especially their mRNA synthesis, was found dependent upon cooperative effects of peptide hormones and heparins (HPs). Comparison of more than 40+ species of intact, native glycosaminoglycans (GAGs) and PGs indicates that only HPs or PGS with HP- like GAG chains, can cooperate with peptide hormones to regulate mRNA synthesis of tissue-specific genes. By contrast, heparin sulfates (HSs), at all concentrations and from all sources, proved inactive or even inhibitory. Posttranscriptional effects were observed with all tested PGs, and of the GAGs, with all species of HPs and weakly with dermantan sulfates (DSs). The rank order of potency of the PGs with respect to posttranscriptional effects was HP-PGs>>HS-PGs>>>DS-PGs>>chondroitin sulfate-PG (CS-PG) and of the active GAGs, HPs>>>>DSs. We propose to complete a structure-function analysis, ongoing, to define the chemistry of active PGs and GAGs enabling them to regulate gene expression. Our completed studies allow us to focus on HPs, the biologically active components of HP-PGs, the most active species of PG able to regulate gene expression in liver cells. HSs are closely related chemically, are inactive, and will be used as negative controls. The biological assays, all documented to be HP sensitive, will be: [1) morphological assays to detect nuclear shape changes;] 2) molecular hybridization assays for mRNA synthesis and abundance of insulin-responsive genes; and 3) electrophysiological assays for gap junctions. Cells will be treated with a test HP or HP fraction when in serum-free medium supplemented only with completely defined and purified hormones or growth factors, the composition and concentrations tailored for each gene. The format of the analysis is to: 1) complete the screens of HPs and of polyanions with HP-like activity (e.g. suramin) allowing us to identify especially active species; 2) dissect the most active ones for contributions of molecular weight (chain length), charge density, degree and form of sulfation, and extent of anticoagulant activity; [3) chemically and structurally analyze the most active fractions of the most active HPs and polyanions; 4) deduce from the chemical analysis what the active fractions have in common and prepare modified versions accordingly; 5) assess the biological activity of the modified versions and 6) repeat 3-5 until the active saccharide sequences (s) are identified.] The most active HP saccharide(s) identified will be used to initiate studies on the signal transduction mechanism(s) involved in HP/insulin's regulation of gene expression. We will focus initially on studies of calcium and intracellular pH, since our prior studies indicated that membrane-permanent analogues of one known second messenger, cAMP, were unable to synergize with HPs in the regulation of transcription rates for any liver-specific gene assayed. [We will also assess whether HPs modify insulin receptor levels or the binding affinity of insulin for its own receptor or for the receptors for IGF I or II.]