MOLECULAR-CLONING OF PIT-1 CDNA FROM PORCINE ANTERIOR-PITUITARY AND ITS INVOLVEMENT IN PITUITARY STIMULATION BY GROWTH HORMONE-RELEASING FACTOR

Pit-1/GHF-1 (hereafter Pit-1) is a pituitary-specific transcription fa ctor that participates in growth hormone (GH), prolactin and thyroid-s timulating hormone gene expression and in proliferation of the cells t hat produce these hormones. In this study, we determined the nucleotid e sequence of porcine Pit-1 cDNA, which shows high homology (95%) with the known mammalian Pit-is. Some differences in amino acid sequence w ere found in the amino terminal region, but the POU-specific and POU-h omeo 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-polymera se chain reaction. Stimulation with GRF for 2 h increased the Pit-1 mR NA level 3-fold. This response was transient, as the level returned to the control level after 6 h. Forskolin and TPA evoked similar increas es, but their effects appeared after 30 min and reached their maxima a fter 2 h. In contrast, GRF and forskolin, but not TPA, increased the G H mRNA level 2-fold after 24 h. The cJun mRNA level showed no signific ant change in response to these agents over 24 h and GRF and TPA incre ased 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 TP A did not appear to be participated straightforward in the GH gene exp ression, suggesting that cFos alone is insufficient to the activation.