Growth hormone (hGH) is known to play an essential role in normal growth and metabolism but has also been associated with the progression of several cancers (e.g. breast, colon, prostate), often linked with increased expression of its receptor (hGHR) and IGF-I. Our research goal is to characterise the mechanism(s) regulating hGHR expression as the first step towards determining what causes its aberrant production in cancer tissues. The hGHR gene has a complex 5’untranslated region (5’UTR): fourteen variant mRNAs are expressed from different 5’UTR exons but all splice into the same site upstream of the translation start site and, thus, code for the same protein. Seven of these exons are clustered within two small regions: V2-V9-V3 (Module A) and V7-V1-V4-V8 (Module B). Module A-derived mRNAs are ubiquitously expressed while those from Module B are expressed in a tissue-, tumor- and developmental-specific fashion (only in normal postnatal liver), indicating that at least two different regulatory mechanisms are involved. One striking difference is that Module A promoters contain response elements (REs) for factors common amongst ubiquitously expressed genes, while Module B contains REs for liver-enriched factors. Secondly, CpG islands surround all three exons of Module A but not Module B. These differential features are conserved in the ovine, bovine, mouse and rat GHR genes.
Studies of proximal promoter regions for Module A exons showed significant activity in HEK293, CV1, HepG2 and Huh7 cell lines. However, when the constructs were partially or fully methylated there was partial or complete loss of promoter activity, respectively. The methylation status of the promoter regions was examined in genomic DNA of two hepatoma cell lines (HepG2, Huh7) as well as human adult and fetal livers. Southern blot analyses for V2 and V9 showed partial methylation in fetal liver (in which hGHR levels are less abundant compared to adult liver), suggesting that expression of the ubiquitous hGHR mRNAs may be regulated by a methylation-specific mechanism.
A second unique feature of Module A is that the promoter regions of each exon have a putative Sp1 binding site. Overexpression of a dominant-negative Sp1 in HEK293 cells showed a significant decrease in promoter activity for all three exons. Parallel experiments in drosophila SL2 cells indicate that overexpression of Sp1 or Sp3 has a stimulatory effect, primarily on a site within the V2 exon. Currently, Sp1 RE deletion and EMSA assays are being done to confirm which Sp1 sites play a significant role in regulating expression of the ubiquitous hGHR mRNAs.
In summary, this study highlights important elements involved in regulating ubiquitous expression of the hGHR and, thus, sensitivity to hGH. This is a first step towards understanding aberrant expression of hGHR in cancer cells.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA