Molecular cloning of pit-1 cDNA from porcine anterior pituitary and its involvement in pituitary stimulation by growth hormone-releasing factor.

Abstract

Pit-1/GHF-1 (hereafter Pit-1) is a pituitary-specific transcription factor that participates in growth hormone (GH), prolactin and thyroid-stimulating hormone gene expression and in proliferation of the cells that produce these hormones. In this study, we determined the nucleotide sequence of porcine Pit-1 cDNA, which shows high homology (95%) with the known mammalian Pit-1s. Some differences in amino acid sequence were found in the amino terminal region, but the POU-specific and POU-homeo domains were well conserved. Porcine anterior pituitary cells in primary culture were treated for 24 h with GH-releasing factor (GRF), forskolin and phorbol ester (TPA). These agents stimulated progressive secretion of GH into the culture media. The levels of Pit-1, GH, cJun and cFos mRNAs were examined using the reverse transcription-polymerase chain reaction. Stimulation with GRF for 2 h increased the Pit-1 mRNA level 3-fold. This response was transient, as the level returned to the control level after 6 h. Forskolin and TPA evoked similar increases, but their effects appeared after 30 min and reached their maxima after 2 h. In contrast, GRF and forskolin, but not TPA, increased the GH mRNA level 2-fold after 24 h. The cJun mRNA level showed no significant change in response to these agents over 24 h and GRF and TPA increased the cFos mRNA level 1.4 and 2.3-fold, respectively, after 30 min. These results suggest that increasing Pit-1 mRNA level in response to GRF plays a role in the early event of the GH gene expression or with the assistance of other transcription factors that modulate GH gene. Furthermore, the GRF-induced increase in cFos mRNA level by GRF and TPA did not appear to be participated straightforward in the GH gene expression, suggesting that cFos alone is insufficient to the activation.