| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
In order to elucidate the molecular mechanisms underlying functional maturation of growth hormone (GH) cells, the mechanisms responsible for the induction of glucocorticoid (GC) receptor expression was investigated using fetal rat pituitary cells in primary culture. The forced expression of glucocorticoid receptor in the pituitary cells from rat fetuses on day 16 of gestation, where no GH expression was detected, resulted in a marked increase in GH expression. In the separate experiment, the immunoreactive GH cells appeared in the similar culture when they were cultured for several days in the medium containing serum. These results suggest that the expression of GC receptor is an important step for the immature GH cells to initiate GH expression and that the factor required for the induction of GC receptor in the fetal GH cells may reside in serum. However, the exact nature of the factor could not be elucidated in this study.
Another result of this study was the elucidation of the molec
… More
ular basis for the GC regulation of the GHRH-receptor gene expression. An approximately 3 kb genomic fragment spanning the promoter region of the gene was cloned, sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. In the presence of dexamethasone (DEX), the GHRH-R promoter (from-11 to-2935) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells. While thyroid hormone, all trans-retinoic acid and 9cis-retinoic acid alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of DEX.Additional analyses of promoter-truncated reporter constructs demonstrated a functional retinoic acid response element in the region between-1167 and-664 and three candidate sequences for a glucocorticoid response element and a thyroid hormone response element in an 80 bp 5'-flanking sequence of the pit-1 site.
These results taken as a whole indicate that GC is an key molecule for the functional maturation of GH cells in the fetal rat pituitary gland, that induces expressions of two GH cell specific molecules, GH and GHRH-receptor. However, the present results showed that the mechanisms for the induction of each molecule are totally different. GC-induction of GH expression is mediated by unknown protein that is induced by GC, no direct interaction between GC receptors and GH promoter being required in this case, while GC acts as the transcription factor to interact with particular sequences in the 5'-upstream region to induce GHRH-receptor expression. Less
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