Biochemical characterization of diverse Stat5b-binding enhancers that mediate growth hormone-activated insulin-like growth factor-I gene transcription.

Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH

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Stat5b binding sites are required to confer GH-responsiveness to <i>Igf1</i> promoter 2 in promoter-reporter assays.

<p>Results of luciferase assays in Cos-7 cells transiently transfected with reporter plasmids containing <i>Igf1</i> P2 and exon 2, plus wild type (WT) or mutated versions of individual Stat5b binding elements, and expression plasmids encoding the mouse GH receptor and rat Stat5b, and incubated with vehicle (dark bars) or rat GH [40 nM] (light bars) for 18 h. KO signifies knockout of a Stat5b binding site, with DKO representing double knockout and TKO, triple knockout. See ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050278#s2" target="_blank">Materials and Methods</a>’ for details. Bars represent the mean ± S.E. of 4–10 independent experiments (*, p<0.007; **, p<0.0007; #, p<0.017; ##, p<0.0017; ^∧, p<0.013 <i>vs.</i> WT with GH [unpaired t-test]). In each graph, relative luciferase values obtained using the WT Stat5b element in the absence of GH were set to 1. <b>A</b>. R2–4. <b>B</b>. R13. <b>C</b>. R34–35. <b>D</b>. R53–54. <b>E</b>. R57–59. <b>F</b>. R60–61.</p

Stat5b binding elements in the rat <i>Igf1</i> locus confer GH-responsiveness to <i>Igf1</i> promoter 2 in promoter-reporter assays. A

<p>. Diagram of the rat <i>Igf1</i> locus showing seven conserved Stat5b binding elements, R2–4, R8–9, R13, R34–35, R53–54, R57–59, and R60–61. Each element is depicted as a gray oval and the six <i>Igf1</i> exons are shown as black boxes. <b>B</b>. <u>Top</u> - schematic of luciferase reporter plasmids containing rat <i>Igf1</i> promoter 2 (P2) and exon 2, and individual Stat5b binding elements (Enhancer). <u>Bottom</u> - Results of luciferase activity assays in Cos-7 cells transiently transfected with expression plasmids encoding the mouse GH receptor and rat Stat5b and reporter plasmids containing each putative enhancer region diagramed to the <u>left</u> (with each Stat5b site indicated as a white oval, and R13.5 as a gray curved shape), after incubation of cells with vehicle (dark bars) or rat GH [40 nM] (light bars) for 18 h. The graph presents results of 4 independent experiments for each promoter plasmid (mean ± S.E.; *, p<0.01; **, p<0.001 <i>vs.</i> R34–35 without GH [unpaired t-test]). Other p values are indicated, and compare ± GH treatment [paired t-test]. Luciferase counts for R34–35 without GH ranged from 3.5 to 5.5×10³ relative light units/sec.</p