Abstract
A key agent in the anabolic actions of growth hormone (GH) is insulin-like growth factor-I (IGF-I), a 70-amino acid secreted protein with direct effects on somatic growth and tissue maintenance and repair. GH rapidly and potently stimulates IGF-I gene transcription by mechanisms independent of new protein synthesis, and recent studies have linked the transcription factor Stat5b to a regulatory network connecting the activated GH receptor on the cell membrane to the IGF-I gene in the nucleus. Here we analyze two distinct conserved GH response elements in the rat IGF-I locus that contain paired Stat5b sites. Each response element binds Stat5b in vivo in a GH-dependent way, as assessed by chromatin immunoprecipitation assays, and consists of one high affinity and one lower affinity Stat5b site, as determined by both qualitative and quantitative protein-DNA binding studies. In biochemical reconstitution experiments, both response elements are able to mediate GH-stimulated and Stat5b-dependent transcription when fused to a reporter gene containing either the major IGF-I promoter or a minimal neutral promoter, although the paired Stat5b sites located in the second IGF-I intron were more than twice as effective as the response element that mapped approximately 73 kb 5' to the IGF-I exon 1. Taken together, our results define the initial molecular architecture of a complicated GH-regulated transcriptional pathway, and suggest that apparently redundant hormone response elements provide a mechanism for amplifying GH action at a physiologically important target gene.
Highlights
The growth hormone (GH)-insulin-like growth factor-I (IGF-I) pathway plays a central role in the control of postnatal somatic growth in humans and in most other mammals (4 – 6)
Recently has experimental evidence emerged demonstrating that Stat5b is a key intermediate in the steps between binding of GH to its cell membrane-spanning receptor and onset of IGF-I gene transcription in the nucleus [11]
By a combination of approaches we previously identified and analyzed a DNA segment termed HS7 in the second intron of the rat IGF-I gene that contains tandem Stat5 binding sites, and showed that it functions as a cis-regulatory region capable of mediating GHinduced IGF-I gene transcription [16]
Summary
Materials—Antibodies to Stat (C17X) were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-FLAG (M2) was from Sigma, and anti-T7 from Novagen (San Diego, CA). Recombinant rat GH was purchased from the National Hormone and Pituitary Program, NIDDK, 3190 JOURNAL OF BIOLOGICAL CHEMISTRY
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