Pituitary tumorigenesis targeted by the ovine follicle-stimulating hormone beta-subunit gene regulatory region in transgenic mice.

Targeted tumorigenesis in transgenic mice has been a powerful tool for the study of gene expression and oncogenesis, as well as for the production of differentiated immortal cell lines from rare cell types. Follicle-stimulating hormone (FSH) is secreted by the gonadotrope cells of the anterior pituitary gland and plays a pivotal role in mammalian reproduction. Here we have used the regulatory region of the ovine FSH beta gene to direct expression of the SV40 T antigen oncogene to gonadotrope cells in the pituitary of transgenic mice. Two of five transgenic mouse lines bearing this fusion gene rapidly developed pituitary tumors, with appearance of adenomatous foci as early as 6 weeks of age, resulting in death by 12 weeks of age in both genders. Histologic examination of tumor development over time revealed that increases in cell proliferation and dysplasia were accompanied by decreases in synthesis of pituitary hormones, indicating dedifferentiation of the pituitary cells. Histological features observed in these tumors were in agreement with this rapid transformation of cell phenotype. Tumors were multifocal in origin, and the most highly transformed cell types observed consisted of giant pale basophilic cells with enormous hyperploid nuclei associated with infiltrating neuronal-like cells, which were very abundant at later stages of tumor development. Mitotic indices were much higher in transgenic than wild-type pituitaries, as expected. Morphologic analysis of the gonads of these transgenic mice showed no major developmental differences, as compared to wild-type littermates, however the length of the seminiferous tubules in transgenic males was greater than age-matched wild-type animals. Despite this phenotype difference, both genders were fertile, with normal sperm development observed in males and normal estrous cycle stages in females. Moreover, while 8 -- 10-week-old transgenic males had much lower blood levels of FSH than littermates, transgenic female FSH levels were the same as those of wild-type females. These animals offer a unique and potentially useful model of organ-specific tumorigenesis, where a multistage pathway of tumor development is evident, both histologically and temporally. Study of such models will advance our knowledge on the physiological and molecular mechanisms involved in gene expression as well as tumor formation.

An Otx-related homeodomain protein binds an LHbeta promoter element important for activation during gonadotrope maturation.

The hormone-secreting cell types of the anterior pituitary differentiate in a specific spatial and temporal manner. The alpha-subunit of the glycoprotein hormones appears at embryonic d 11.5 in the mouse, followed by steroidogenic factor-1, which distinguishes the gonadotrope progenitor cells, around embryonic d 14. Gonadotrope maturation is marked by the onset of LHbeta-gene expression 2 d later. The alphaT3-1 and LbetaT2 immortalized mouse pituitary cell lines correspond to these later sequential stages of gonadotrope differentiation. In addition to the early markers of the gonadotrope lineage present in alphaT3-1 cells, LbetaT2 cells also express markers of a mature gonadotrope, including LHbeta and FSHbeta. Using transient transfections to compare expression among gonadotrope and nongonadotrope-derived cell types, we show that the rat 1.8-kb LHbeta promoter directs reporter gene expression specifically to the mature gonadotrope LbetaT2 cell line. Promoter truncation and mutagenesis analyses indicate that the homeodomain (HD) element located at approximately -100 bp relative to the transcriptional start site is essential for this selectivity to LbetaT2 cells when compared with alphaT3-1 cells. In EMSAs, this HD site binds a protein present in LbetaT2 but not other gonadotrope-derived cells. Antibody supershift and competition experiments indicate that this LbetaT2 nuclear protein is a K50 HD protein related to the Otx family, though it is not a known pituitary homeobox transcription factor protein. These studies indicate a role for a novel Otx-related HD protein in gonadotrope maturation during development.

Transcriptional activation of the ovine follicle-stimulating hormone-beta gene by gonadotropin-releasing hormone involves multiple signal transduction pathways.

GnRH regulates gonadotrope cells through GnRH receptor activation of the PKC-, MAPK-, and calcium-activated signaling cascades. Due to the paucity of homologous model systems expressing FSHbeta, little is known about the specific mechanisms involved in transcriptional regulation of this gene by GnRH. Previous studies from our laboratory demonstrated that the gonadotrope-derived LbetaT2 cell line expresses FSHbeta mRNA. In the present study we characterized the mechanisms involved in GnRH regulation of the FSHbeta promoter using this cell model. Using transfection assays, we show that GnRH regulation of the ovine FSHbeta promoter involves at least two elements, present between -4152/-2878 and -2550/-1089 bp, in association with one or several elements within the proximal region of the promoter. Surprisingly, the two activating protein-1 sites previously shown to be involved in the FSHbeta response to GnRH in heterologous cells do not play a role in GnRH responsiveness in the gonadotrope cell model. Here we demonstrate that calcium influx itself is not sufficient to confer the response, but it is necessary for both 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and GnRH induction of the FSHbeta gene. Moreover, we show that GnRH regulation of FSHbeta gene expression is mediated by PKC and establish the presence of multiple PKC isozymes in LbetaT2 cells. Interestingly, GnRH and TPA induce activity of the FSHbeta promoter through different, although possibly overlapping, pools of PKC isoforms. This is further supported by the use of a MAPK inhibitor, which abolishes the induction of FSHbeta by GnRH, but not by TPA. In conclusion, we have demonstrated that calcium, PKC, and MAPK signaling systems are all involved in the induction of FSHbeta gene expression by GnRH in the LbetaT2 mouse gonadotrope cell model.