@article{huang_wu_su_zhirnov_miller_2001, title={A novel role for bone morphogenetic proteins in the synthesis of follicle-stimulating hormone}, volume={142}, ISSN={["1945-7170"]}, DOI={10.1210/en.142.6.2275}, abstractNote={FSH is produced in pituitary gonadotropes as an α/β heterodimer, and synthesis of the β-subunit is the rate-limiting step in overall FSH production. Synthesis of FSHβ can be regulated by activin and inhibin, both of which are members of the transforming growth factor-β superfamily. Bone morphogenetic proteins (BMPs) also belong to the transforming growth factor-β family and are multifunctional growth factors involved in many aspects of tissue development and morphogenesis, including regulation of FSH action in the ovary. Here we report a novel function for BMP-7 and BMP-6 in regulating FSH synthesis in the pituitary. Using primary pituitary cell cultures derived from transgenic mice that carry the ovine FSHβ promoter linked to a luciferase reporter gene (oFSHβLuc), BMP-7 or BMP-6 was found to stimulate oFSHβLuc expression by 6-fold. Transient expression of the oFSHβLuc in a transformed gonadotrope cell line, LβT2, was induced 4-fold by BMP-7 or BMP-6 treatment. More importantly, BMP-7 and BMP-6 increased endogenous FSH secretion by 10- and 14-fold, respectively, from LβT2 cells, demonstrating for the first time that a functional signaling BMP system is present in gonadotropes. Two bioneutralizing antibodies to BMP-7, which cross-react with BMP-6, but not with activin A, decreased basal oFSHβLuc expression and FSH secretion from transgenic mouse pituitary cultures by 83–88% and 47–48%, respectively, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH synthesis. Neither bioneutralizing antibody to activin A or activin B decreased basal oFSHβLuc expression or mouse FSH secretion significantly. Dose-dependent inhibition of FSH synthesis by anti-BMP7 was also observed in rat and sheep pituitary cultures. These results combined with the fact that the messenger RNAs for BMP-7 and BMP-6 were detected in mouse pituitaries and LβT2 cells indicate that BMP-7 and/or BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression in vivo.}, number={6}, journal={ENDOCRINOLOGY}, author={Huang, HJ and Wu, JC and Su, P and Zhirnov, O and Miller, WL}, year={2001}, month={Jun}, pages={2275–2283} }
 @article{pernasetti_vasilyev_rosenberg_bailey_huang_miller_mellon_2001, title={Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the L beta T2 pituitary gonadotrope cell model}, volume={142}, ISSN={["0013-7227"]}, DOI={10.1210/en.142.6.2284}, abstractNote={FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LβT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the α- and β-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH β- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHβ 5′-regulatory region (5.5 kb) confers LβT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHβ regulatory region responds to activin specifically in LβT2 cells, an effect that is blocked by follistatin. The LHβ, α-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LβT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHβ gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHβ and LHβ regulatory regions, specifically in LβT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LβT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GnRH.}, number={6}, journal={ENDOCRINOLOGY}, author={Pernasetti, F and Vasilyev, VV and Rosenberg, SB and Bailey, JS and Huang, HJ and Miller, WL and Mellon, PL}, year={2001}, month={Jun}, pages={2284–2295} }
 @article{huang_sebastian_strahl_wu_miller_2001, title={The promoter for the ovine follicle-stimulating hormone-beta gene (FSH beta) confers FSH beta-like expression on luciferase in transgenic mice: Regulatory studies in vivo and in vitro}, volume={142}, ISSN={["0013-7227"]}, DOI={10.1210/en.142.6.2260}, abstractNote={Transgenic mice harboring the ovine FSHβ (oFSHβ) promoter plus first intron (from −4741 to +759 bp) linked to a luciferase reporter gene (oFSHβLuc) were generated to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for studying hormonal regulation of the FSHβ gene. Of six lines of transgenic mice analyzed, luciferase was detected uniquely in the pituitaries of five of them. Pituitary luciferase activity was decreased 51–99% by chronic GnRH treatment (Lupron depot). Orchidectomy caused a 2- to 8-fold increase, and ovariectomy caused a 2- to 27-fold increase in pituitary luciferase activity. Furthermore, pituitary luciferase expression was consistently higher on estrus than on diestrus (3- to 20-fold). These data strongly suggested that the transgene was expressed specifically in pituitary gonadotropes and regulated in the same way as the endogenous mouse FSHβ gene. Using primary pituitary cell cultures prepared from these transgenic mice, basal luciferase expression was maximal on day 3 and then decreased by day 6 of culture, a pattern reflected by endogenous mouse FSH secretion. In these pituitary cultures, basal oFSHβLuc expression was decreased 61–82% by follistatin or 59–79% by inhibin. Similarly, mouse FSH secretion was decreased 71% by follistatin or 65% by inhibin. Progesterone inhibited oFSHβLuc expression by 44–51%, but it had no effect on endogenous mouse FSH secretion. Estradiol lowered FSH secretion by 21%, but did not decrease oFSHβLuc expression significantly. In conclusion, these data demonstrated the ability of the oFSHβ promoter to direct expression of a reporter gene specifically to pituitary gonadotropes in transgenic mice. Studying oFSHβLuc expression in vivo and in cell cultures derived from pituitaries of these transgenic mice should prove useful for understanding many features of FSHβ regulation.}, number={6}, journal={ENDOCRINOLOGY}, author={Huang, HJ and Sebastian, J and Strahl, BD and Wu, JC and Miller, WL}, year={2001}, month={Jun}, pages={2260–2266} }
 @article{huang_sebastian_strahl_wu_miller_2001, title={Transcriptional regulation of the ovine follicle-stimulating hormone-beta gene by activin and gonadotropin-releasing hormone (GnRH): Involvement of two proximal activator protein-1 sites for GnRH stimulation}, volume={142}, ISSN={["0013-7227"]}, DOI={10.1210/en.142.6.2267}, abstractNote={Previous studies from our laboratory demonstrated that a transgene consisting of the promoter for the ovine FSH β-subunit gene and a luciferase reporter (wt-oFSHβLuc) was expressed and regulated like the FSHβ gene in vivo and in vitro. In the present study pituitary cultures were prepared from these transgenic mice as well as mice carrying mutated oFSHβLuc lacking two functional activator protein-1 (AP-1) sites at −120 and −83 bp (mut-oFSHβLuc). These AP-1 sites were reported necessary for induction of oFSHβLuc by GnRH in a HeLa cell system. To examine the importance of the two AP-1 sites in mediating GnRH and activin effects in primary gonadotropes, pituitary cultures derived from transgenic mice were pretreated with follistatin to remove activin or activin-like factors present in the cultures. Follistatin lowered luciferase expression in cultures carrying both wt-oFSHβLuc and mut-oFSHβLuc transgenes by 74–86%, and subsequent addition of activin induced luciferase expression of both wt- and mut-transgenes by 4- to 14-fold within 4 h, suggesting that these AP-1 sites are not involved in activin stimulation of FSHβ gene transcription. When GnRH was added along with activin, the wt-oFSHβLuc transgene was induced 200% compared with activin alone, but this effect was not observed with the mut-oFSHβLuc transgene. These data confirmed the HeLa cell studies showing that GnRH signals through two AP-1 sites to increase oFSHβ transcription in gonadotropes. However, as the mutation of both AP-1 sites had no apparent effect on the expression and regulation of the transgene in vivo (basal, castration, GnRH down-regulation, cycle stage, and GnRH immunoneutralization), it appears that these AP-1 sites have little influence over the major effect of GnRH observed in vivo. These data also showed that activin plays a major role in transcriptional regulation of the FSHβ gene, and the oFSHβ promoter contains the activin response element(s) that is as yet undefined.}, number={6}, journal={ENDOCRINOLOGY}, author={Huang, HJ and Sebastian, J and Strahl, BD and Wu, JC and Miller, WL}, year={2001}, month={Jun}, pages={2267–2274} }
 @article{strahl_huang_sebastian_ghosh_miller_1998, title={Transcriptional activation of the ovine follicle-stimulating hormone beta-subunit gene by gonadotropin-releasing hormone: Involvement of two activating protein-1-binding sites and protein kinase C}, volume={139}, ISSN={["1945-7170"]}, DOI={10.1210/en.139.11.4455}, number={11}, journal={ENDOCRINOLOGY}, author={Strahl, BD and Huang, HJ and Sebastian, J and Ghosh, BR and Miller, WL}, year={1998}, month={Nov}, pages={4455–4465} }
 @article{strahl_huang_pedersen_wu_ghosh_miller_1997, title={Two proximal activating protein-1-binding sites are sufficient to stimulate transcription of the ovine follicle-stimulating hormone-beta gene}, volume={138}, ISSN={["1945-7170"]}, DOI={10.1210/en.138.6.2621}, abstractNote={FSH is an important regulator of mammalian gametogenesis and the female reproductive cycle. Although little is known about the transcriptional regulation of the β-subunit (the rate-limiting subunit of FSH synthesis), sequence analysis of the ovine FSHβ promoter has revealed a number of potential activating protein-1 (AP-1; Jun/Fos)-binding sites. To determine whether the gene encoding theβ -subunit of ovine FSH (oFSHβ) is responsive to AP-1 transcriptional complexes, chimeric constructs containing deleted portions of the oFSHβ promoter fused to a luciferase reporter were transiently transfected along with c-Jun and c-Fos expression constructs into JAR cells. Analysis of these deletion constructs revealed that the proximal promoter of oFSHβ is highly stimulated by c-Jun and c-Fos proteins (typically 20-fold with a reporter construct containing oFSHβ sequences from −215 to +759 bp). This stimulation was lost when a similar construct containing sequences from −84 to+ 759 bp was tested. Transcriptional start site analysis using reverse transcription-PCR verified that the transcriptional initiation of the− 215-bp deletion construct, with or without cotransfected c-Jun/c-Fos, was the same as that observed in vivo. Computer analysis of oFSHβ sequences from −215 to +1 bp identified four putative AP-1-like elements, located at −155, −120, −83, and −10 bp. Gel retardation experiments using oligonucleotides corresponding to the four putative AP-1-like sites revealed that only −120 and −83 sites in oFSHβ bound AP-1 proteins in vitro. Site-directed mutagenesis of the −120 and −83 sites showed that each element was required for stimulation by c-Jun and c-Fos proteins as well as 12-O-tetradecanoyl phorbol-13-acetate in transient transfection assays. Finally, immunocytochemical dual labeling was used to show that more than 75% of all FSHβ-containing cells in ovine pituitary sections from cycling ewes contained nuclear c-Jun, JunB, JunD, and Fos proteins. These data, taken together, show that oFSHβ transcription can be stimulated by c-Jun and c-Fos proteins via two functionally linked AP-1-like sites in the oFSHβ proximal promoter and that these sites are likely to be important regulators of FSH production in vivo.}, number={6}, journal={ENDOCRINOLOGY}, author={Strahl, BD and Huang, HJ and Pedersen, NR and Wu, JC and Ghosh, BR and Miller, WL}, year={1997}, month={Jun}, pages={2621–2631} }