Margaret A. Shupnik

Luteinizing hormone (LH) is synthesized and secreted throughout the reproductive cycle from gonadotrope cells in the anterior pituitary, and is required for steroidogenesis and ovulation. LH contains an α-subunit common with FSH, and a unique LHβ subunit that defines biological activity. Basal LHβ transcription is low and stimulated by hypothalamic GnRH, which induces synthesis of early growth response protein-1 (Egr1), and stimulates binding of transcription factors Egr1 and steroidogenic factor-1 (SF1) on the promoter. WT1 (Wilms tumor protein1) is a zinc finger transcription factor with an essential role in urogenital system development, and which regulates several reproductive genes via interactions with SF1 or binding to GC-rich elements such as Egr1 binding sites. We investigated a potential role for WT1 in LHβ transcription in clonal mouse gonadotrope LβT2 cells. WT1 was present in LβT2 and mouse pituitary cells, and protein bound to the endogenous LHβ promoter. Interestingly, mRNAs for WT1(+KTS), which contains a three amino-acid insertion between the 3^rd and 4^th zinc fingers, and the WT1 (-KTS) variant were both expressed at significant levels. WT1 mRNAs and protein were decreased approximately 50% by GnRH treatment, under conditions where Egr1 mRNA and protein, and LHβ transcription, were stimulated. Decreasing expression of mRNA for WT1 (-KTS) decreased stimulation of LHβ and Egr1 by GnRH, whereas decreasing both WT1 (-KTS) and (+KTS) increased endogenous LHβ transcription, and prevented LHβ but not Egr1 stimulation by GnRH, suggesting differing biological activities for the WT1 isoforms. Overexpression of WT1 showed that WT1(-KTS) enhanced LHβ promoter GnRH stimulation 2-to-3-fold and required the 3’Egr1 site, but WT1(+KTS) repressed both basal and GnRH-stimulated LHβ promoter activity by approximately 70%. Our data suggest that WT1 can modulate LHβ transcription, with differential roles for the two WT1 variants; WT1 (-KTS) enhances and WT1 (+KTS) suppresses transcription.

GnRH regulation of pituitary gonadotropin gene transcription is critical for fertility, and metabolic dysregulation is associated with reproductive disorders and altered hypothalamic-pituitary responses. Here, we examined signaling pathways in gonadotropes through which GnRH modulates gonadotropin levels, and potential common signaling pathways with insulin. Using LβT2 cells, we show that GnRH rapidly (5 minutes) triggers activating phosphorylation of AMP-activated protein kinase (AMPK) up to 5-fold; this stimulation is enhanced by insulin through increased total AMPKα levels and activity. GnRH also stimulated c-Jun N-terminal kinase (JNK) and ERK activation, whereas insulin alone stimulated Akt. Inhibition of AMPK activity by compound C, or diminishing AMPK levels by small interfering RNA against AMPKα, prevented GnRH-stimulated transcription of the endogenous LHβ gene and transfected LHβ promoter. Egr-1 (early growth response-1), a transcription factor required for LHβ expression, is synthesized in response to GnRH, and compound C prevents this induction. However, overexpression of Egr-1 in the presence of compound C did not restore GnRH stimulation of LHβ, suggesting that AMPK stimulation of transcription also occurs through additional mechanisms or signaling pathways. One such pathway may be JNK activation, because GnRH stimulation of JNK activity and LHβ transcription occurs more slowly than stimulation of AMPK activity, and AMPK inhibition by compound C or small interfering RNA also prevented GnRH-stimulated JNK phosphorylation. Finally, in primary mouse pituitary cells, GnRH also stimulates AMPK, and AMPK inhibition suppresses GnRH-stimulated LHβ transcription. These studies indicate a novel role for AMPK in GnRH-stimulated transcription in pituitary gonadotropes and a potential common mechanism for GnRH and metabolic modulation of fertility.

This year, in response to member input and suggestions to highlight the vibrant basic science of endocrinology, The Endocrine Society Annual Meeting (ENDO 08) introduced a new feature, The Year in Basic Science series. Among the many interests and strengths of our members, three broad areas were chosen for initial consideration: nuclear receptors, kinase signaling, and hormones and cancer. Speakers were invited to present and discuss important publications during the past year between annual meetings (roughly June to June), and to put these findings into broad perspective for the endocrine community. Three distinguished researchers, Bert O'Malley, Tony Means, and Kate Horwitz, graciously agreed to participate in the inaugural venture, and this series of articles is based on their presentations at ENDO 08. Each individual approached this somewhat daunting task slightly differently. However, all three observed important and often common themes that ultimately link current basic molecular findings to broad translational and clinical problems, including metabolism and energy balance, neuronal migration and synapse formation, long-term memory formation, and endocrine pathology in cancer, reproduction and osteoporosis.

The breast cancer susceptibility gene 1 (BRCA1) is mutated in approximately 50% of hereditary breast cancers, and its expression is decreased in 30–40% of sporadic breast cancers, suggesting a general role in breast cancer development. BRCA1 physically and functionally interacts with estrogen receptor-α (ERα) and several transcriptional regulators. We investigated the relationship between cellular BRCA1 levels and tamoxifen sensitivity. Decreasing BRCA1 expression in breast cancer cells by small interfering RNA alleviated tamoxifen-mediated growth inhibition and abolished tamoxifen suppression of several endogenous ER-targeted genes. ER-stimulated transcription and cytoplasmic signaling was increased without detectable changes in ER or ER coregulator expression. Co-immunoprecipitation studies showed that with BRCA1 knockdown, tamoxifen-bound ERα was inappropriately associated with coactivators, and not effectively with corepressors. Chromatin immunoprecipitation studies demonstrated that with tamoxifen, BRCA1 knockdown did not change ERα promoter occupancy, but resulted in increased coactivator and decreased corepressor recruitment onto the endogenous cyclin D1 promoter. Our results suggest that decreased BRCA1 levels modify ERα-mediated transcription and regulation of cell proliferation in part by altering ERα–coregulator association. In the presence of tamoxifen, decreased BRCA1 expression results in increased coactivator and decreased corepressor recruitment on ER-regulated gene promoters.

In the previous issue of the journal, Lin and coworkers present data demonstrate that increased expression of estrogen receptor (ER)-β in ER-α-positive breast cancer cells antagonizes a defined group of ER-α/estrogen stimulated genes that are involved in cell cycle regulation and DNA replication. Similar expression patterns for these genes were found human ER-α positive breast tumors expressing higher levels or ER-β, and this correlated with better clinical outcome. The implications for these data, which suggest that ER-β is a positive actor and diagnostic marker for therapeutic outcome, are discussed.

Both steroids and growth factors stimulate proliferation of steroid-dependent tumor cells, and interaction between these signaling pathways may occur at several levels. Steroid receptors are typically classified as ligand-activated transcription factors, and steps by which they bind ligand, dimerize, recruit coregulatory molecules, and activate target gene transcription are well understood. Several steroid responses are functionally linked to c-Src or tyrosine kinase receptors, and the physiological impact and the precise molecular pathways involved in these responses are under intensive investigation. Ligand-independent stimulation of steroid receptor-mediated transcription by growth factors is now believed to occur through activated protein kinases that phosphorylate the receptors and receptor coregulators. Recently, steroid hormones themselves have been shown to rapidly activate intracellular signaling cascades, via binding to cognate cytoplasmic or membrane-associated receptors. In some contexts, steroid receptors interact directly with c-Src and other cytoplasmic signaling molecules, such as Shc, PI3K, and p130 Cas. Crosstalk between growth factors and steroids in both the cytoplasm and nucleus could have profound impact on complex biological processes such as cell growth, and play a significant role in the treatment of steroid-dependent cancers. The potential roles of progesterone and estrogen receptors in this crosstalk are discussed in this review.

Information on oestrogen action has grown exponentially in the past decade, and recent studies have begun to define the mechanism of ligand‐dependent activation and cell‐specific effects. Oestrogen‐mediated gene transcription in a specific tissue depends on several factors, the most important of which is the presence of at least one of the two nuclear oestrogen receptor (ER) isoforms, ERα and ERβ. The presence and levels of specific ER isoform variants, along with receptor coactivator, corepressor and integrator proteins, directly modulate overall nuclear ER activity. The structure of the ligand, including both physiological oestrogens and synthetic oestrogen receptor modulators, influences ER interactions with these other proteins and thus determines the biological response. Furthermore, peptide and neurotransmitter‐stimulated intracellular signalling pathways activate specific enzyme cascades and may modify the receptors and their interacting proteins, resulting in either independent or ligand‐enhanced ER‐mediated responses. Finally, several rapid effects of oestrogen probably occur at the membrane through nongenomic pathways that may or may not require the same ER proteins that are found in the nucleus. This review concentrates on the pituitary‐hypothalamic axis and the genomic effects of oestrogen, and discusses the current knowledge of each of these factors in determining oestrogen actions in the neuroendocrine system.

T3 suppression of TSH subunit gene transcription is an important step in maintaining thyroid hormone homeostasis, and recent investigations have increased our understanding of this process. Thyrotrope-specific proteins play a critical role in TSH subunit gene expression, and influence T3-mediated regulatory mechanisms. The structure and placement of the TSH gene TREs define suppressive regulation by T3, and this process is favored by the TR isoforms expressed in the pituitary. Elimination of TR beta function compromises the pituitary response to T3. TR beta 2, the isoform specifically expressed in pituitary and neural tissue, contains a transferable domain that both increases T3-independent gene transcription and enhances T3-suppressed transcription. The functional interaction of TR beta 2 with other regulatory proteins is distinct from that of other TR isoforms, and likely plays a critical role in pituitary physiology and in pituitary resistance to thyroid hormone. The development of novel thyrotrope cell lines will allow investigators to define new proteins and molecular mechanisms that distinguish negative from positive T3 transcriptional regulation.