Activin markedly stimulates FSH β messenger RNA (mRNA) levels in rat pituitary cells. Nonetheless, the molecular mechanisms through which activin enhances FSH β gene expression are not clear. To assess the role of transcriptional activation in activin stimulation, we first transfected two -2300FSH β Luc constructs into primary pituitary cells and treated them with activin in plated culture and perifusion. Basal expression of the constructs was low, and no activin response was observed. These results suggested that additional FSH β sequences are required for basal expression or that the effects of activin are not transcriptional. An alternative approach for measuring transcriptional responses was developed based upon changes in the levels of FSH β primary transcripts (FSH β-PT; newly transcribed mRNAs that still contain the first intron) after activin (3 ng/ml) stimulation of perifused rat pituitary cells. An increase in FSH β-PT was observed after 30 min of activin stimulation, preceding the first observable rise in mature FSH β mRNA. Levels of FSH β-PT peaked between 1-2 h, then fell to a lower level (28% of maximal) at 4 h, which was maintained through 10 h of activin stimulation (36% of maximal at 10 h). Mature FSH β mRNA levels peaked between 2-4 h, which is after the increase in FSH β-PT, and fell more gradually between 4-10 h of stimulation (56% of maximal at 10 h). Unstimulated levels of mature mRNA and FSH β-PT did not vary significantly over the course of the experiments. Cotreatment with the transcriptional inhibitor actinomycin-D (2 microM) blocked activin stimulation of both FSH β-PT and FSH β mRNA, confirming the transcriptional basis for these events. In summary, we have documented rapid and sequential increases in FSH β-PT and mature FSH β mRNAs after activin stimulation, which are prevented by transcriptional blockade. These data provide evidence that the increase in FSH β mRNA levels after activin treatment are at least partly due to transcriptional activation of the FSH β gene.
ASJC Scopus subject areas