Transcript abundance in mouse pituitaries with altered growth hormone expression quantified by reverse transcriptase polymerase chain reaction implicates transcription factor Zn-16 in gene regulation in vivo.
The correlation of growth hormone (GH) mRNA abundance and expression of specific transcription factors was studied in pituitaries of panhypopituitary (Ames df/df and Snell dwJ/dwJ dwarf), isolated GH-deficient (lit/lit), and GH-overproducing (growth hormone-releasing hormone [GHRH] transgenic) mice compared with normal littermates. A fluorescence-based reverse transcriptase polymerase chain reaction assay was developed for seven target mRNAs: GH, prolactin (PRL), pro-opiomelanocortin (POMC), alpha-subunit of the glycoprotein hormones (alphaSU), Pit-1, Prop-1, and Zn-16. Amplification parameters for each of these primer pairs were determined in order to calculate initial mRNA transcript number. The reproducibility of the assay was found to be +/-10% for either Pit-1 or Zn-16 mRNAs measured in characterized murine GHFT1-5 somatotroph precursor cells. The cell extracts also showed an increased abundance of both Zn-16 and Pit-1 mRNAs when compared with whole pituitary extracts. Measurement of copy number in normal pituitaries showed that for every 10(6) GH or PRL mRNAs, there were 3 x 10(5) POMC, 4 x 10(4) alphaSU, 2 x 10(3) Pit-1, and only 70 Zn-16 or Prop-1 transcripts. Transcript abundance in GH-altered mice as a percentage of copy number per normal gland showed that POMC was significantly reduced in dwJ/dwJ (p < 0.01) and df/df (p < 0.05) mice. AlphaSU mRNA was reduced in df/df (p < 0.05), dwJ/dwJ (p < 0.05), and lit/lit (p < 0.05) mice, but not in GHRH-excess mice. PRL mRNA was not detected in dwarf mice, reduced to 52% of normal in lit/lit (p < 0.05), and unchanged in GHRH-excess animals. GH mRNA was not detected in dwarf mice, reduced to 1.3% in lit/lit (p < 0.005), and increased to 242% in GHRH-excess mice (p < 0.05). Pit-1 mRNA was not detected in dwarf mice, was 2.9% of normal in lit/lit (p < 0.005) mice, and increased to 200% in GHRH-excess mice (p < 0.05). Prop-1 was not present in dwarf mice, was decreased to 1.4% in lit/lit (p < 0.01), and increased to 223% in GHRH-excess mice (p < 0.05). Zn-16 abundance in df/df mice was significantly reduced (p < 0.05) to 4.8% of normals, to 6.3% of normals in dwJ/dwJ (p < 0.005), to 6.1% of normals in lit/lit (p < 0.005) mice, and significantly elevated in GHRH-excess mice to 197% (p < 0.05). Altered pituitary mRNA abundance was found for several products not previously measured, or thought not to be affected by these mutations. Correlation of GH mRNA abundance with transcription factor copy number showed a significant correlation for Pit-1, Prop-1, and Zn-16. These quantitative analyses provide the first in vivo evidence that Zn-16 mRNA abundance correlates with GH expression.
Wojtkiewicz
Patrick W
PW
Molecular and Cellular Biology Program, Tulane University, New Orleans, LA, USA.
Phelps
Carol J
CJ
Hurley
David L
DL
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D004393:Dwarfism, Pituitary / Q000378:metabolism; D005786:Gene Expression Regulation; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics; D013006:Growth Hormone / Q000235:genetics*; D013007:Growth Hormone-Releasing Hormone / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D011333:Pro-Opiomelanocortin / Q000235:genetics; D011388:Prolactin / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics*
D004268:DNA-Binding Proteins; D015292:Glycoprotein Hormones, alpha Subunit; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone; D018398:Homeodomain Proteins; D011333:Pro-Opiomelanocortin; D011388:Prolactin; D012333:RNA, Messenger; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494185:Pou1f1 protein, mouse; C102134:Prophet of Pit-1 protein; C406423:Zfp292 protein, mouse