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.]
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