Search for regulatory factors of the pituitary-specific transcription factor PROP1 gene.

Pituitary-specific transcription factor PROP1, a factor important for pituitary organogenesis, appears on rat embryonic day 11.5 (E11.5) in SOX2-expressing stem/progenitor cells and always coexists with SOX2 throughout life. PROP1-positive cells at one point occupy all cells in Rathke's pouch, followed by a rapid decrease in their number. Their regulatory factors, except for RBP-J, have not yet been clarified. This study aimed to use the 3 kb upstream region and 1st intron of mouse prop1 to pinpoint a group of factors selected on the basis of expression in the early pituitary gland for expression of Prop1. Reporter assays for SOX2 and RBP-J showed that the stem/progenitor marker SOX2 has cell type-dependent inhibitory and activating functions through the proximal and distal upstream regions of Prop1, respectively, while RBP-J had small regulatory activity in some cell lines. Reporter assays for another 39 factors using the 3 kb upstream regions in CHO cells ultimately revealed that 8 factors, MSX2, PAX6, PIT1, PITX1, PITX2, RPF1, SOX8 and SOX11, but not RBP-J, regulate Prop1 expression. Furthermore, a synergy effect with SOX2 was observed for an additional 10 factors, FOXJ1, HES1, HEY1, HEY2, KLF6, MSX1, RUNX1, TEAD2, YBX2 and ZFP36Ll, which did not show substantial independent action. Thus, we demonstrated 19 candidates, including SOX2, to be regulatory factors of Prop1 expression.

Expression of Krüppel-like factor 6, KLF6, in rat pituitary stem/progenitor cells and its regulation of the PRRX2 gene.

Paired-related transcription factors, PRRX1 and PRRX2, which are present in mesenchymal tissues and participate in mesenchymal cell differentiation, were recently found in the stem/progenitor cells of the pituitary gland of ectodermal origin. To clarify the role of PRRX1 and PRRX2 in the pituitary gland, the present study first aimed to identify transcription factors that regulate Prrx1 and Prrx2 expression. A promoter assay for the upstream regions of both genes was performed by co-transfection of the expression vector of several transcription factors, many of which are frequently found in the pituitary stem/progenitor cells. The results for the promoter activity of both genes showed expression in a cell type-dependent manner. Comprehensive comparison of transcriptional activity of several transcription factors was performed with CHO cells, which do not show Prrx1 and Prrx2 expression, and the results revealed the presence of common and distinct factors for both genes. Among them, KLF6 showed specific and remarkable stimulation of Prrx2 expression. In vitro experiments using an electrophoretic mobility shift assay and siRNA interference revealed a potential ability for regulation of Prrx2 expression by KLF6. Finally, immunohistochemistry confirmed the presence of KLF6 in the SOX2/PRRX2 double-positive stem/progenitor cells of the postnatal pituitary gland. Thus, the finding of KLF6 might provide a novel clue to clarify the maintenance of stem/progenitor cells of the postnatal pituitary gland.

Molecular cloning of rat and porcine retina-derived POU domain factor 1 (POU6F2) from a pituitary cDNA library.

Homeobox transcription factors are known to play crucial roles in the anterior lobe of the pituitary gland. During molecular cloning with the Yeast One-Hybrid System using a 5'-upstream region of the porcine Fshß as a bait sequence, we have cloned a cDNA encoding a partial sequence of the retina-derived POU domain factor 1 (RPF1) from the porcine pituitary cDNA library and confirmed its specific binding to the bait sequence. In situ hybridization was performed to examine localization of Rpf1 and showed that this gene is expressed in the stem/progenitor cells of the rat pituitary primordium as well as the diencephalon and retina. In addition, real-time PCR demonstrated that Rpf1 transcripts are abundant in early embryonic periods but that this is followed by a decrease during pituitary development, indicating that this factor plays a role in differentiating cells of the pituitary. The transcriptional activity of RPF1 for genes of Prop1, Prrx1 and Prrx2, which were characterized as genes participating in the pituitary stem/progenitor cells by our group, was then examined with full-length cDNA obtained from the rat pituitary. RPF1 showed regulatory activity for Prop1 and Prrx2, but not for Prrx1. These results indicate the involvement of this retina-derived factor in pituitary development.

GFP-expressing S100ß-positive cells of the rat anterior pituitary differentiate into hormone-producing cells.

Some non-endocrine cells in the pituitary anterior lobe are responsible for providing stem/progenitor cells to maintain hormone-producing cells. In particular, cells expressing S100ß protein, a calcium-binding protein, have been hypothesized to be a pituitary cell resource. Accumulating data have revealed that S100ß-positive cells comprise heterogeneous populations and some of them certainly show stem/progenitor characteristics in vivo. Hence, we examine whether S100ß-positive cells have the capacity to differentiate into endocrine cells, by means of in vivo and in vitro experiments on transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of the S100ß promoter. Immunohistochemistry of the pituitary confirmed that some S100ß-positive cells expressed SOX2 (SRY [sex-determining region Y]-box 2) and had proliferative activity. Dispersed anterior lobe cells were observed by time-lapse microscopy, followed by immunostaining for hormone and pituitary-transcription-factor1 (PIT1). First, the dispersed anterior lobe cells were immunostained by an antibody against SOX2. S100ß-protein co-localizes with SOX2 (about 89 %). Although 44 of 134 S100ß-positive cells traced were proliferative but negative to any hormones, 14 cells were positive for one of the pituitary hormones and/or PIT1, confirming the presence of all types of hormone-producing cells. Notably, GFP-fluorescence appeared in two hormone-positive cells during culture. On the other hand, we observed hormone-producing cells that were not positive for S100ß at the end of the time-lapse study, despite being initially positive. These findings suggest that S100ß-positive cells cultured from the anterior lobe are capable of developing into hormone-producing cells, although this happens relatively infrequently.